CA3111978A1 - Chimeric antigen receptor for solid cancer and t cells expressing chimeric antigen receptor - Google Patents
Chimeric antigen receptor for solid cancer and t cells expressing chimeric antigen receptor Download PDFInfo
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- CA3111978A1 CA3111978A1 CA3111978A CA3111978A CA3111978A1 CA 3111978 A1 CA3111978 A1 CA 3111978A1 CA 3111978 A CA3111978 A CA 3111978A CA 3111978 A CA3111978 A CA 3111978A CA 3111978 A1 CA3111978 A1 CA 3111978A1
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Abstract
Disclosed is a chimeric antigen receptor with improved persistency.
Description
[DESCRIPTION]
[Invention Title]
CHIMERIC ANTIGEN RECEPTOR FOR SOLID CANCER AND T CELLS
EXPRESSING CHIMERIC ANTIGEN RECEPTOR
[Technical Field]
The present invention relates to a novel chimeric antigen receptor (CAR) for effectively treating blood cancer or solid cancer, and to a CAR-T cell in which a chimeric antigen receptor targeting a specific cancer antigen is expressed. In addition, the present invention relates to a vector which expresses a novel CAR for effectively treating blood cancer or solid cancer.
More specifically, the present invention relates to a chimeric antigen receptor comprising an antigen binding domain; a hinge region; a transmembrane domain;
a costimulatory domain; and a cytoplasmic signaling domain, in which any one immunoreceptor tyrosine-based activation motif (hereinafter described as "ITAM") present within a CD3 domain, which is a cytoplasmic signaling domain, is mutated for excellent in vivo persistence and anti-tumor effects of CAR-T cells, and to a CAR-T cell in which the chimeric antigen receptor is expressed.
Further, in order to induce a cytokine signal in an antigen-dependent manner during stimulation of cancer antigens, a cleaved cytoplasmic domain of interleukin-7 receptor-a (IL7Ra) or interleukin-2 receptor-p (IL-2R[3) in addition to the CD3 signaling domain and a 4-1BB costimulatory domain was added to a CAR gene so as to reduce the differentiation and exhaustion of CAR-T cells caused by CAR tonic signaling of CAR-T cells, thereby making it capable of exhibiting excellent in vivo persistence and anti-tumor effects.
In addition, in the present invention, a chimeric switch receptor was introduced to convert the immunosuppressive signal in the hostile tumor microenvironment into an activation signal in CAR-T cells.
In addition, in the present invention, the cytokine IL-21 is released out of CAR-T
cells so as to aid the activation of innate immune-related cells and increase the content of less-differentiated memory CAR-T cells.
The chimeric antigen receptor according to the present invention can show excellent in vivo persistence and anti-tumor effects of CAR-T cells in a hostile tumor microenvironment, and can simultaneously reduce toxicity, thereby reducing side effects in the patient while improving the treatment effect. In addition, it is possible to expand the scope of use of the present invention by preparing a chimeric antigen receptor-provided allogenic (non-self-derived) CAR-T treatment according to the present invention.
[Background]
T cells expressing chimeric antigen receptor (CAR) (CAR-T cells) refers to a type of T cell with a recombinant gene, in which a gene encoding a receptor that recognizes cancer cell surface antigens specifically expressed on the surface of cancer cells is Date Recue/Date Received 2021-03-05 1 introduced into a T cell to kill cancer cells.
Dr. Zelig Eshhar, an Israeli chemist and immunologist at the Weizmann Institute of Science, et al. previously proposed a hypothesis that when T cells are artificially made with a receptor that binds to an antigen specifically expressed in a cancer cell, it is possible to target only cancer cells and trigger an immune response to kill cancer cells. They succeeded in preparing T cells provided with a chimeric antigen receptor, and this was reported in PNAS in 1989.
In the CAR-T cells prepared in the early days (i.e., the first-generation CAR-T
cells), only CD3 was used as a signaling domain. However, these CAR-T cells had drawbacks in that the treatment effect was insignificant and that the persistence was also short. Therefore, efforts have been made to improve the reactivity of CAR-T cells, and as a result, second-generation CAR-T cells were prepared in which a costimulatory domain (0D28 or 0D137/4-1BB) and CD3 were combined, and these CAR-T cells showed an increase in the number of CAR-T cells remaining in the body compared to the first-generation CAR-T cells.
In the second-generation CAR-T cells, only one kind of costimulatory domain was used, and CAR-T cells using two kinds of costimulatory domains are referred to as third-generation CAR-T.
With regard to the method of treating cancer using CAR-T cells, three late-stage chronic lymphoid leukemia (CCL) patients were injected with cytotoxic T cells which were modified to recognize CD19. As a result, leukemia was completely cured in two of the patients, and there were reports that the conditions lasted for about 10 months (N
Engl J Med 2011, 365:725-733, August 25, 2011; Sic Transl Med 2011 Aug 10, 3(95):95ra73). In particular, the CAR-T used corresponded to the second-generation CAR-T cells, in which 4-1BB was used as the costimulatory domain and CD3 was used as the signaling domain. The antigen binding domain of the CAR-T cells recognizes CD19 found on the surface of leukemia cancer cells as an antigen.
In addition, there was a report that when CTL019 was administered to patients with acute leukemia, 27 out of 30 patients experienced complete remission, 67%
of the patients experienced complete remission for over 2 years, and 78% of the patients survived for two years. Considering that the target patients above were relapsed or refractory patients, these results were very surprising (N Engl J Med 2014, 371:1507-1517, October 16, 2014).
Currently, clinical trials for various blood cancers (e.g., lymphoma, myeloma, etc.) are in progress with regard to therapeutic methods using various CAR-T cells, and CAR-T, as a pharmaceutical drug that can be used for the treatment of blood cancer, has entered the market.
However, there are several obstacles to using CAR-T cells for the treatment of solid cancer. For example, when CAR-T cells are administered intravenously to a cancer patient, it is difficult for CAR-T cells to migrate to the tumor site;
and in a hostile tumor microenvironment, CAR-T cells are functionally inhibited while simultaneously exhibiting limited persistence and proliferation.
Therefore, there is a need to develop CAR-T cells which can treat solid cancer by overcoming these problems.
Date Recue/Date Received 2021-03-05 2 [Detailed Description of the Invention]
[Technical Problem]
An object of the present invention is to provide CAR platforms, CAR-T cells, and CAR expression vectors which have remarkably excellent therapeutic effects in solid cancer compared to the previously known CAR-T cells.
The CAR-T cell therapeutic of the present invention for treating solid cancer comprises: (1) a mutated ITAM introduced into the cytoplasmic signaling domain for excellent in vivo persistence and antitumor effects of CAR-T cells; and/or (2) CAR tonic signaling controlled by adding a cleaved cytoplasmic domain of IL7Ra or IL-2R[3 in addition to a CD3 signaling domain and a 4-1BB costimulatory domain to a CAR
gene so as to induce a cytokine signal in an antigen-dependent manner; and/or (3) a chimeric switch receptor introduced so as to convert the immunosuppressive signal in the hostile tumor microenvironment into an activation signal in CAR-T cells; and/or (4) cytokine IL-21 allowed to release out of CAR-T cells so as to aid the activation of innate immune-related cells and increase the content of less-differentiated memory CAR-T
cells.
Due to CAR tonic signaling, CAR-T cells promote anergy, differentiation, exhaustion, and activation-induced CAR-T cell death. In order to increase the therapeutic efficacy of CAR-T cells for treating solid cancer, excellent in vivo persistence and anti-tumor effects of CAR-T cells are essential. Therefore, it is necessary to control the CAR tonic signaling system in strategies for solid cancer treatment.
Since the CAR tonic signaling can be controlled by substitution of a cytosolic signaling domain of CAR-T cell, an antigen-dependent cytokine signal was added into CAR-T cell. Additionally, in order to control the CAR tonic signaling that may occur due to uncontrolled excessive ITAM-based signaling within the CD3 domain, one mutated ITAM was introduced into the cytoplasmic signaling domain. By controlling CAR
tonic signaling, the excellent in vivo persistence and anti-tumor effects of CAR-T
cells were made to last longer.
In addition, a chimeric switch receptor was introduced, which converts the inhibitory signal of TGF-r3 (i.e., an immunosuppressive cytokine overexpressed in a hostile tumor microenvironment) into an activation signal in CAR-T cells. In addition, it was prepared such that cytokine IL-21 could be released out of CAR-T cells so as to aid the activation of innate immune-related cells and increase the content of less-differentiated memory CAR-T cells.
The chimeric antigen receptor of the present invention can be used to prepare CAR-T cell therapeutics for various carcinomas by changing the cancer antigen target in the antigen binding domain.
[Technical Solution]
To solve the problems described above, the present invention provides a polypeptide comprising a chimeric antigen receptor, which comprises an antigen binding domain; a hinge region; a transmembrane domain; a costimulatory domain; and a signaling domain. In particular, the costimulatory domain may comprise a 4-1BB
Date Recue/Date Received 2021-03-05 3 domain or 0D28 domain or both a 4-1BB domain and a 0D28 domain, in which the signaling domain comprises a CD3 domain.
In the present invention, the polypeptide comprises IL-7Ra or a part thereof interposed between the 4-1BB domain and the CD3 domain. In particular, a part of IL-7Ra may be a sequence of SEQ ID NO: 15, and a part of IL-21R8 may be a sequence of SEQ ID NO: 17. A part of the cytoplasmic domain of the IL-7Ra (SEQ ID NO: 15) is a sequence between the 266th and the 328th positions in the entire sequence of IL-7Ra of SEQ ID NO: 14, and a part of the cytoplasmic domain of the IL-21R8 (SEQ ID NO:
17) is a sequence between the 266th and the 369th positions in the entire sequence of of SEQ ID NO: 16.
For example, in vivo persistence and anti-tumor effects of CAR-T cells were increased by controlling the CAR tonic signaling by linking a cleaved cytoplasmic domain of IL-7Ra (amino acids at positions from 265 to 328; SEQ ID NO: 15) or cleaved cytoplasmic domain of IL2R8 (amino acids at positions from 266 to 369; SEQ ID
NO: 17) between 4-1BB (SEQ ID NO: 11) as a costimulatory domain and mutant CD3 (in which the positions from 91 to 113 of SEQ ID NO: 18 are substituted with either SEQ
ID
NO: 31 or SEQ ID NO: 32), in a CAR which is specific to IL-13Ra2, based on the known anti-IL13Ra2 CAR-T cells (YYB-103 of SEQ ID NO: 22: see PCT International Publication No. WO 2017/023138).
In the present invention, three immunoreceptor tyrosine-based activation motifs (ITAMs) are included within the CD3 domain, and in a third ITAM region among these, it is preferred that a first motif, YxxL, and a second motif, YxxL, be substituted (in which x and y each represent any amino acid) (see FIG. 3).
Generally, the ITAM within the CD3 domain includes two forms of YxxL (or isoleucine (I)) containing tyrosine (Y), which is spaced apart from leucine (L) or isoleucine (I) by a distance of a two amino acid sequence. Since the YxxL/I
forms are usually present to be spaced apart by 6 to 8 amino acids, the ITAM can be represented by the structure of YxxL/Ix(6_8)Y)o(L/1.
In the present invention, three ITAMs (positions 21 to 35; positions 60 to 75;
and positions 91 to 105 of SEQ ID NO: 18) are included within the CD3 domain, which is represented by SEQ ID NO: 18, in which in the CD3 domain, it is preferred that in a third ITAM region, a first motif, YxxL (positions 91 to 94 of SEQ ID NO: 18), be substituted with YxxQ, and a second motif region, YxxLHM (positions 102 to 107 of SEQ ID NO: 18), be substituted with YxYVTM, or in a third ITAM region, a first motif, YxxL (positions 91 to 94 of SEQ ID NO: 18), be substituted with YyyL, and a second motif, YxxL (positions 102 to 105 of SEQ ID NO: 18), be substituted with YxxQ
(in which x and y each represent any amino acid).
For example, in the present invention, the region including a third ITAM
region of the CD3 domain may be mutated to a sequence of SEQ ID NO: 31 or 32.
Specifically, a chimeric antigen receptor (CAR) can be used, in which among the three ITAM motifs present within the CD3 domain (see SEQ ID NO: 18) (i.e., a known signaling domain of anti-IL13Ra2 CAR-T cells (YYB-103)), the sequence of a third ITAM
motif (i.e., positions 91 to 106 of SEQ ID NO: 18) is mutated from "YQGLSTATKDTYDALHMQALPPR" to "YLPQSTATKDTYDYVTMQALPPR" (SEQ ID
Date Recue/Date Received 2021-03-05 4 NO: 31); or among the three ITAM motifs present within the CD3 domain (see SEQ
ID
NO: 18) (i.e., a known signaling domain of anti-IL13Ra2 CAR-T cells (YYB-103)), the sequence of a third ITAM motif (i.e., positions 91 to 105 of SEQ ID NO: 18) is mutated from "YQGLSTATKDTYDALHMQALPPR" to "YLSLSTATKDTYLPQHMQALPPR" (SEQ
ID NO: 32) (see FIG. 3).
In particular, in the present invention, it is preferred that in a third ITAM
region of the CD3 domain, a first motif, YxxL (positions 91 to 94 of SEQ ID NO: 18), be substituted with YxxQ, and a second motif, YxxLHM (positions 102 to 106 of SEQ
ID
NO: 18), be substituted with YxYVTM, or in a third ITAM region of the CD3 domain, a first motif, YxxL (positions 91 to 94 of SEQ ID NO: 18), be substituted with YyyL, and a second motif, YxxL (positions 102 to 105 of SEQ ID NO: 18), be substituted with YxxQ
(in which x and y each represent any amino acid).
The polypeptide of the present invention may further contain a cytokine. For example, the polypeptide of the present invention may contain IL-21 as a cytokine to be added. In particular, it is preferred that the cytokine be linked to a chimeric antigen receptor by a self-cleaving peptide.
As the self-cleaving peptide, for example, the known 2A peptides of SEQ ID
NOS: 40 to 43 may be used. The self-cleaving peptide undergoes a post-translational cleavage, in which the peptide bond between a proline (P) and a glycine (G) in the C-terminus is digested by cleavage. Therefore, the proteins located upstream and downstream of the self-cleaving peptide are expressed independently of each other.
Accordingly, when the polypeptide according to the present invention is expressed in a T cell, the cytokine can be separated from a chimeric antigen receptor (CAR), and the separated cytokine can be released to the outside of the T
cell. Innate immune-related cells can be activated by the separated cytokine (e.g., IL-21), and thereby the content of less-differentiated CAR-T cells can be increased (see FIG. 5).
That is, in vitro differentiation is prevented due to the IL-21 expression during the process of CAR-T cell preparation, which results in an increase of the content of less-differentiated memory CAR-T cells, thereby making it possible to provide excellent in vivo persistence and enhanced anti-tumor effects of CAR-T cells, and which additionally results in activation of the innate immune cells around the solid cancer, thereby making it possible to more effectively treat solid cancer.
The polypeptide of the present invention may further include a TGF-8R2 exodomain, an IL18R transmembrane domain, and an IL18R endodomain, in addition to a chimeric antigen receptor. In particular, it is preferred that an IL18R
transmembrane domain be included between the TGF-8R2 exodomain and the IL18R endodomain (see FIG. 4).
In the present invention, it is preferred that the TGF-8R2 exodomain be linked to a chimeric antigen receptor by a self-cleaving peptide. Therefore, when the polypeptide according to the present invention is expressed in T cells, the polypeptide comprising a TGF-8R2 exodomain, an IL18R transmembrane domain, and an IL18R endodomain is separated from a chimeric antigen receptor (CAR). The TGF-8R2 domain of the separated polypeptide is exposed to the outside of T cells, the IL18R
transmembrane domain is located in the cell membrane of T cells, and the IL18R endodomain is located Date Recue/Date Received 2021-03-05 5 in the cytoplasm of T cells.
Accordingly, when TGF-8, which is an immunosuppressive cytokine, is present outside of T cells, TGF-8 binds to the TGF-8R2 exodomain, thereby activating the IL18R endodomain. That is, the inhibitory signal of TGF-8 (i.e., an immunosuppressive cytokine in a hostile tumor microenvironment) is converted into an activation signal of the cytokine IL-18 in CAR-T cells. Therefore, a polypeptide, which includes the TGF-8R2 exodomain, IL18R transmembrane domain, and IL18R endodomain, becomes a chimeric switch receptor.
IL-18, which is a cytokine, inhibits expression of immunosuppressive materials in T cells and differentiation of regulatory T cells, and enhances immune responses against cancer cells. When the effects of the CAR-T cell therapeutic by IL-18 in advanced solid tumors were examined, it was confirmed that Fox01 expression in CD4+
T cells was decreased by IL-18, and additionally that T-bet expression was increased by IL-18 in CD8+ T cells.
Ultimately, IL-18 shows an excellent anticancer effect in advanced solid cancer through the T-bethigh Fox01low CAR-T, persistent CAR-T cells. In particular, in the present invention, a chimeric switch receptor, which includes a TGF-8R2 exodomain (amino acid positions of 23 to 166; SEQ ID NO: 19), which is a receptor for binding to TGF-8 (i.e., an immunosuppressive cytokine around solid cancer); and a transmembrane domain and an endodomain of IL-18R (amino acid positions of 323 to 541; SEQ ID NO: 20), which is a cytokine receptor, was prepared to be expressed in CAR-T cells, thereby changing the immunosuppressive signal into an activation signal in CAR-T cells by making a reverse use of the hostile tumor microenvironment.
As described above, even a polypeptide, which additionally includes a TGF-8R2 exodomain, an IL18R transmembrane domain, and an IL18R endodomain in the chimeric antigen receptor, may further include the cytokine described above (e.g., IL-21).
That is, the cytokine described above (e.g., IL-21) may be linked by a self-cleaving peptide to the IL18R endodomain of the polypeptide, which additionally includes a TGF-8R2 exodomain, an IL18R transmembrane domain, and an IL18R
cytoplasmic endodomain in the chimeric antigen receptor.
Accordingly, when the polypeptide according to the present invention is expressed in T cells, the polypeptide which includes the TGF-8R2 exodomain, transmembrane domain, and IL18R endodomain can be separated from the chimeric antigen receptor (CAR), and additionally, the cytokine can also be independently separated. Therefore, the separated cytokine can be released to the outside of T cells, the TGF-8R2 exodomain is exposed to the outside of the T cells, the IL18R
transmembrane domain is located in the cell membrane of T cells, and the IL18R
endodomain is located in the cytoplasm of T cells.
The polypeptide according to the present invention may be, for example, a polypeptide represented by any one of SEQ ID NOS: 24 to 30 and 34 to 37.
In addition, the present invention relates to a CAR-T cell in which the CAR-containing polypeptide is expressed as described above.
In addition, the present invention relates to a chimeric antigen receptor (CAR) expression vector, which includes an antigen binding domain; a hinge region; a Date Recue/Date Received 2021-03-05 6 transmembrane domain; a costimulatory domain; and a cytoplasmic signaling domain.
In particular, the CAR expression vector includes a nucleic acid encoding a CAR, in which the nucleic acid encoding a CAR comprises a nucleic acid encoding a 4-domain as the costimulatory domain and a nucleic acid encoding a CD3 domain as the signaling domain, and further comprises a nucleic acid encoding IL-7Ra or a part thereof or a nucleic acid encoding IL-2Rp or a part thereof between the nucleic acid encoding the 4-1BB domain and the nucleic acid encoding the CD3 domain. In particular, a part of IL-7Ra may be, for example, a sequence of SEQ ID NO: 15, and a part of IL-2Rp may be, for example, a sequence of SEQ ID NO: 17.
In the present invention, the nucleic acid encoding the CD3 domain may be a nucleic acid encoding a CD3 domain, in which in a third ITAM region among the three ITAMs present within the CD3 domain, a first motif, YxxL, and a second motif, YxxLHM, are substituted. Preferably, the nucleic acid encoding the CD3 domain may be a nucleic acid encoding a CD3 domain, in which in a third ITAM region, a first motif, YxxL, is substituted with YxxQ, and a second motif region, YxxLHM, is substituted with YxYVTM, or one in which a first motif, YxxL, is substituted with YyyL, and a second motif region, YxxL, is substituted with YxxQ (in which x and y each represent any amino acid).
In addition, the CAR expression vector of the present invention may further include a nucleic acid encoding cytokine IL-21. In particular, the nucleic acid encoding cytokine IL-21 is linked to a nucleic acid encoding a CAR through a nucleic acid encoding a self-cleaving peptide.
In addition, the CAR expression vector of the present invention may further include a nucleic acid encoding a TGF-pR2 exodomain, a nucleic acid encoding an IL18R transmembrane domain, and a nucleic acid encoding an IL18R endodomain.
In particular, the nucleic acid encoding the TGF-pR2 exodomain is linked to a nucleic acid encoding a CAR through a nucleic acid encoding a self-cleaving peptide.
As described above, the CAR expression vector, which includes a nucleic acid encoding a TGF-pR2 exodomain, a nucleic acid encoding an IL18R transmembrane domain, and a nucleic acid encoding an IL18R endodomain, may further include a nucleic acid encoding cytokine IL-21. In particular, the nucleic acid encoding cytokine IL-21 is linked to a nucleic acid encoding an IL18R endodomain through a nucleic acid encoding a self-cleaving peptide.
The present invention relates to a CAR-T cell prepared by introducing the CAR
expression vector described above.
In addition, the present invention relates to an anticancer agent containing the CAR-T cell described above. The anticancer agent of the present invention containing the CAR-T cell may further contain a pharmaceutically acceptable additive as necessary.
In the CAR-containing polypeptides according to the present invention, CAR-T
cells for various carcinomas may be prepared by changing the cancer antigen target of the antigen binding domain as necessary. For example, the antigen binding domain may be prepared as those which bind to IL13Ra2, an antigen associated with an angiogenesis activity (anti-angiogenesis), and antigens such as EGFRvIll, EphA2, aVpMesothelin, and glypican1. That is, if any ligand or antibody targeting IL-13Ra2, Date Recue/Date Received 2021-03-05 7 anti-angiogenesis, EGFRvIll, EphA2, aVpglypican1, and mesothelin (see SEQ ID
NOS: 1 to 7 and 33) is introduced, these CAR-T cells may be used as an anticancer agent for these targets. Therefore, it is possible to prepare CAR-T cells having an anti-cancer effect against a specific carcinoma.
In the case of EGFRvIll (i.e., a major tumor antigen expressed in glioblastoma, lung cancer, etc.), in order to reduce side effects of CAR-T cells that appear through a non-specific binding, a CAR expression rate and persistence in CAR-T cells were optimized while simultaneously changing the target sequence so as to minimize the binding affinity with EGFR wild-type while maintaining the specificity for EGFRvIll.
While changing positions 52 to 57 of SEQ ID NO: 3 from STGGYN to DPENDE
(a CDR2 part of a heavy chain), position 101 of SEQ ID NO: 3 from S to G (a CDR3 part of a heavy chain), and position 229 of SEQ ID NO: 3 from V to G (a CDR3 part of a light chain) in the target sequence (SEQ ID NO: 33), a CAR expression rate and persistence in CAR-T cells were improved using CD8 signal sequence (SEQ ID NOS: 34 and 35).
In order to increase a CAR expression rate and persistence in CAR-T cells targeting aV[3, a Gaussia princeps luciferase signal sequence or CD8 signal sequence was used (SEQ ID NOS: 36 and 37).
[Advantageous Effects]
The CAR-T cells disclosed in the present invention have an excellent expression rate and persistence; therefore, these cells show the effects of persistence and anti-tumor effects in the human body, thereby having an improved therapeutic effect against solid cancer.
[Brief Description of Drawings]
FIG. 1 shows a diagram illustrating a process of effectively treating solid cancer by a CAR-containing polypeptide according to the present invention.
FIG. 2 shows a diagram and a table illustrating an antigen binding domain which can be used for a CAR-containing polypeptide platform according to the present invention.
FIG. 3 shows diagrams illustrating the introduction of a cytokine signaling domain into CAR-T cells so as to control CAR tonic signaling in the CAR-containing polypeptide according to the present invention.
FIG. 4 shows diagrams illustrating the introduction of a chimeric switch receptor so as to convert an immune suppressive signal in a hostile tumor microenvironment into an activation signal in CAR-T cells.
FIG. 5 shows diagrams illustrating the release of cytokine IL-21 to the outside of CAR-T cells so as to aid the activation of innate immune-related cells and to increase the content of less-differentiated memory CAR-T cells.
FIG. 6 shows diagrams illustrating the structures of CAR-containing polypeptides according to the present invention.
FIG. 7 shows diagrams illustrating the self-cleaving peptides used in the CAR-containing polypeptides according to the present invention.
FIG. 8 shows a graph and tables illustrating the growth rate and viability of CAR-Date Recue/Date Received 2021-03-05 8 T cells, which are transformed with the CAR of the present invention.
FIG. 9 shows graphs and a table illustrating the analysis results of the CAR
expression rate of CAR-T cells, which were transformed with the CAR of the present invention, by flow cytometry analysis.
FIG. 10 shows a graph and a table illustrating the analysis results of the chimeric switch receptor expression rate of TGF-8R2 and IL-18R of CAR-T cells, which were transformed with the CAR of the present invention, by flow cytometry analysis.
FIG. 11 shows the results of Day 10 phenotypes of CAR-T cells, which were transformed with the CAR of the present invention, by flow cytometry analysis.
FIG. 12 shows a graph, which illustrates the cytotoxicity of CAR-T cells, which were transformed with the CAR of the present invention, confirmed through LDH
assay after 24 hours of co-culture with U87 cells (human brain cancer cell line) and target cells (which were used as a control group for normal cells); and a table, which illustrates the purity and viability of CAR-T cells used in a CAR-T
cytotoxicity test.
FIG. 13 shows graphs illustrating the analysis results of spontaneous toxicity of CAR-T cells, which were transformed with the CAR of the present invention, after 24 hours or 96 hours.
FIGS. 14 to 16 each show graphs and a table illustrating the analysis results of changes in a CAR expression rate of CAR-T cells, which were transformed with the CAR of the present invention, after 24 hours (FIG. 14), 48 hours (FIG. 15), and 96 hours (FIG. 16) of co-culture of the CAR-T cells with U87 cells (human brain cancer cell line) and 293FT target cells (which were used as a control group for normal cells), by flow cytometry analysis.
FIG. 17 shows graphs and a table illustrating the analysis results of changes in the chimeric switch receptor expression rate of TGF-8R2 and IL-18R of CAR-T
cells, which were transformed with the CAR of the present invention, after 24 hours of co-culture of the CAR-T cells, which were transformed with the CAR of the present invention, with U87 cells (human brain cancer cell line) and 293FT target cells (which were used as a control group for normal cells), by flow cytometry analysis.
FIG. 18 shows graphs illustrating the comparison results of IFN-y cytokine production after 24 hours and 48 hours of co-culture of CAR-T cells, which were transformed with the CAR of the present invention, with U87 cells (human brain cancer cell line) and 293FT target cells (which were used as a control group for normal cells).
FIG. 19 shows graphs illustrating the comparison results of IL-21 cytokine production after 24 hours and 48 hours of co-culture of CAR-T cells, which were transformed with the CAR of the present invention, with U87 cells (human brain cancer cell line) and 293FT target cells (which were used as a control group for normal cells).
FIG. 20 shows a graph and tables illustrating the growth rate and viability of CAR-T cells, which were transformed with a chimeric antigen receptor (CAR), when the cancer antigen is EGFRvIll, in which the CAR expression rate and persistence in CAR-T cells were optimized, while simultaneously changing the target sequence so as to minimize the binding affinity for EGFR wild-type, where the specificity for EGFRvIll is maintained so as to reduce the side effects of CAR-T cells that appear through non-specific binding (YYB105, #13, #14), and a graph and tables illustrating the growth rate Date Recue/Date Received 2021-03-05 9 and viability of CAR-T cells, which were transformed with a chimeric antigen receptor (CAR), when the cancer antigen is aV[3, in which the CAR expression rate in CAR-T
cells was optimized (YYB107, #15, #16).
FIG. 21 shows a graph illustrating the CAR expression rate in CAR-T cells, which were transformed with a chimeric antigen receptor (CAR), when the cancer antigen is EGFRvIll, in which the CAR expression rate in CAR-T cells was optimized, while simultaneously changing the target sequence so as to minimize the binding affinity for EGFR wild-type, where the specificity for EGFRvIll is maintained so as to reduce the side effects of CAR-T cells that appear through non-specific binding (YYB105, #13, #14), and a graph illustrating the CAR expression rate in CAR-T cells, which were transformed with a chimeric antigen receptor (CAR), when the cancer antigen is aV[3, in which the CAR expression rate in CAR-T cells was optimized (YYB107, #15, #16).
[Best Mode for Carrying Out the Invention]
Hereinafter, the present invention will be described in detail through examples.
However, it should be noted that the following Examples are only for illustrating the present invention, and the spirit and technical scope of the present invention are not limited in any sense. Further, it should be understood that the present invention is not limited to the precise arrangement and means of the embodiments shown in the drawings, which are cited as reference.
Example 1: Preparation of novel chimeric antigen receptor (CAR) platform for effective treatment by specifically binding to IL13Ra2 overexpressed in solid cancer cells With regard to human IL13 (P35225.1), human CD3 (P20963-1), human CD8A
(P01732), human 0D28 (P10747), human CD3 (P20963), human 41BB (Q07011), IL7RA (P16871), IL2RB (P14784), TGFR2 (P37173), IL18R (Q13478), IL21 (Q9HBE4), IL2 (P60568), T2A, P2A, and human kappa light chain signal sequence (HuVHCAMP), CAR-containing polypeptides were prepared using a chimeric antigen-containing polypeptide (see SEQ ID NOS: 23 to 30 and 34 to 37; and #1, #2, #5, #6, #7, #8, #11, and #12 of FIG. 6) expression vector consisting of codon-optimized synthetic DNA, using scientific literature and publicly available databases (see FIGS. 6 and 7).
Specifically, T cells, in which a CAR-containing polypeptide is expressed, can be prepared by preparing a vector that expresses a CAR-containing polypeptide via conjugation of synthetic DNA to an MFG retrovirus expression vector digested with Xhol/Notl, followed by transduction of the vector into the T cells.
The completed structure of polypeptide #1 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human IL13.E11K.R64D.S67D.R107K mature protein sequence (see YYB-103 of PCT
International Publication No. WO 2017/023138), three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein (see YYB-103 of PCT International Publication No. WO 2017/023138), a hinge region of human CD8a, a human CD8 transmembrane domain, a costimulatory signal domain of Date Recue/Date Received 2021-03-05 10 cytoplasmic 4-1BB, a cleaved cytoplasmic domain of cytokine IL-7RA (amino acid positions of 265 to 328; SEQ ID NO: 15), a mutant CD3 (positions 91 to 113 of SEQ ID
NO: 18 are mutated to a sequence of SEQ ID NO: 31), T2A, a CD8A signal sequence (leader sequence), a chimeric switch receptor which comprises TGF-8R2 exodomain (amino acid positions of 23 to 166; SEQ ID NO: 19) and a transmembrane domain and an endodomain of IL-18R (i.e., a cytokine receptor) (amino acid positions of 323 to 541;
SEQ ID NO: 20), P2A, a human 1L2 signal sequence (leader sequence), an IL-21 (amino acid positions of 23 to 155; SEQ ID NO: 21) sequence, and an Xhol/Notl cleavage site (see CAR-containing polypeptide #1 of FIG. 6; and FIG. 7).
The completed structure of CAR-containing polypeptide #2 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human 11_13.E11K.R64D.S67D.R107K mature protein sequence, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8a, a human CD8 transmembrane domain, a costimulatory signal domain of cytoplasmic 4-1BB, a cleaved cytoplasmic domain of a cytokine IL-7RA (amino acid positions of 265 to 328; SEQ ID NO: 15), a mutant CD3 (positions 91 to 113 of SEQ ID NO: 18 are mutated to a sequence of SEQ ID NO: 31), T2A, a CD8A signal sequence (leader sequence), a chimeric switch receptor which comprises TGF-8R2 exodomain (amino acid positions of 23 to 166; SEQ ID NO: 19) and a transmembrane domain and an endodomain of IL-18R (i.e., a cytokine receptor) (amino acid positions of 323 to 541;
SEQ ID NO: 20), and an Xhol/Notl cleavage site (see CAR-containing polypeptide #2 of FIG. 6; and FIG. 7).
The completed structure of CAR-containing polypeptide #5 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human 11_13.E11K.R64D.S67D.R107K mature protein sequence, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8a, a human CD8 transmembrane domain, a costimulatory signal domain of cytoplasmic 4-1BB, a cleaved cytoplasmic domain of a cytokine IL-7RA (amino acid positions of 265 to 328; SEQ ID NO: 15), a mutant CD3 (positions 91 to 113 of SEQ ID NO: 18 are mutated to a sequence of SEQ ID NO: 31), T2A, a human 1L2 signal sequence, an (amino acid positions of 23 to 155; SEQ ID NO: 21) sequence, and an Xhol/Notl cleavage site (see CAR-containing polypeptide #5 of FIG. 6; and FIG. 7).
The completed structure of CAR-containing polypeptide #6 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human 11_13.E11K.R64D.S67D.R107K mature protein sequence, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8a, a human CD8 transmembrane domain, a costimulatory signal domain of cytoplasmic 4-1BB, a cytoplasmic domain of a cytokine IL-7RA (amino acid positions of 265 to 328;
Date Recue/Date Received 2021-03-05 11 SEQ ID NO: 15), a mutant CD3 (positions 91 to 113 of SEQ ID NO: 18 are mutated to a sequence of SEQ ID NO: 31), and an Xhol/Notl cleavage site (see CAR-containing polypeptide #6 of FIG. 6; and FIG. 7).
The completed structure of polypeptide CAR-containing #7 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human 11_13.E11K.R64D.S67D.R107K mature protein sequence, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8a, a human CD8 transmembrane domain, a costimulatory signal domain of cytoplasmic 4-1BB, a cleaved cytoplasmic domain of a cytokine IL2R8 (amino acid positions of 266 to 369; SEQ ID NO: 17), a mutant CD3 (positions 91 to 113 of SEQ ID NO: 18 are mutated to a sequence of SEQ ID NO: 32), T2A, a CD8A signal sequence, a chimeric switch receptor which comprises a TGF-8R2 exodomain (amino acid positions of 23 to 166; SEQ ID NO: 19) and a transmembrane domain and an endodomain of IL-18R
(i.e., a cytokine receptor) (amino acid positions of 323 to 541; SEQ ID NO: 20), P2A, a human 1L2 signal sequence (leader sequence), an IL-21 (amino acid positions of 23 to 155; SEQ ID NO: 21) sequence, and an Xhol/Notl cleavage site (see #7 of FIG.
6; and FIG. 7).
The completed structure of CAR-containing polypeptide #8 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human 11_13.E11K.R64D.S67D.R107K mature protein sequence, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8a, a human CD8 transmembrane domain, a costimulatory signal domain of cytoplasmic 4-1BB, a cleaved cytoplasmic domain of a cytokine IL2R8 (amino acid positions of 266 to 369; SEQ ID NO: 19), a mutant CD3 (positions 91 to 113 of SEQ ID NO: 18 are mutated to a sequence of SEQ ID NO: 32), T2A, a CD8A signal sequence, a chimeric switch receptor which comprises a TGF-8R2 exodomain (amino acid positions of 23 to 166; SEQ ID NO: 19) and a transmembrane domain and an endodomain of IL-18R
(i.e., a cytokine receptor) (amino acid positions of 323 to 541; SEQ ID NO: 20), and an Xhol/Notl cleavage site (see CAR-containing polypeptide #8 of FIG. 6; and FIG.
7).
The completed structure of CAR-containing polypeptide #11 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human 11_13.E11K.R64D.S67D.R107K mature protein sequence, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8a, a human CD8 transmembrane domain, a costimulatory signal domain of cytoplasmic 4-1BB, a cleaved cytoplasmic domain of a cytokine IL2R8 (amino acid positions of 266 to 369; SEQ ID NO: 17), a mutant CD3 (positions 91 to 113 of SEQ ID NO: 18 are mutated to a sequence of SEQ ID NO: 32), T2A, a human 1L2 signal sequence, an (amino acid positions of 23 to 155; SEQ ID NO: 21) sequence, and an Xhol/Notl Date Recue/Date Received 2021-03-05 12 cleavage site (see CAR-containing polypeptide #11 of FIG. 6; and FIG. 7).
The completed structure of polypeptide CAR-containing #12 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human 11_13.E11K.R64D.S67D.R107K mature protein sequence, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a human hinge region of CD8a, a human CD8 transmembrane domain, a costimulatory signal domain of cytoplasmic 4-1BB, a cleaved cytoplasmic domain of a cytokine IL2R8 (amino acid positions of 266 to 369; SEQ ID NO: 17), a mutant CD3 (positions 91 to 113 of SEQ ID NO: 18 are mutated to a sequence of SEQ ID NO: 32), and an Xhol/Notl cleavage site (see #12 of FIG. 6; and FIG. 7).
The entire sequences of CAR-containing polypeptides #1, #2, #5, #6, #7, #8, #11, and #12 are shown in SEQ ID NOS: 23 to 30.
The finally prepared CAR gene fragments were conjugated to the MFG retrovirus expression vector digested with Xhol/Notl (Emtage PC et al., Clin Cancer Res, 2008, 14:8112-8122). In this Example, in order to compare the activity of chimeric antigen receptors, YYB103 (SEQ ID NO: 22) was further prepared.
Example 2: Preparation of CAR-T cells transformed with novel chimeric antigen receptors High-titer CAR-expressing PG13 clones were prepared such that Phoenix-Ampho and Phoenix-Eco cells were transiently transfected with the retroviral expression vector prepared in Example 1, and then, cell-free vector stocks were prepared from the transfected Phoenix-Ampho and Phoenix-Eco cells by transfecting PG13 cells.
For high-titer monoclones, PG13/#1, PG13/#2, PG13/#5, PG13/#6, PG13/#7, PG13/#8, PG13/#11, or PG13/#12 cells were stained using an anti-IL-13 monoclonal antibody (BD Pharmingen), and these single clones were isolated using a flow cytometer. The PG13/#1, PG13/#2, PG13/#5, PG13/#6, PG13/#7, PG13/#8, PG13/#11, or PG13/#12 clones were prepared by the second subcloning according to the limiting dilution method. These subclones stably showed high CAR expression and were selected for the efficient transduction ability in the peripheral blood.
The transduction degree of PG13/#1, PG13/#2, PG13/#5, PG13/#6, PG13/#7, PG13/#8, PG13/#11, or PG13/#12 cells, which were transduced using an anti-IL-monoclonal antibody (BD Pharmingen), was analyzed using a flow cytometer. The supernatants of the transduced PG13/#1, PG13/#2, PG13/#5, PG13/#6, PG13/#7, PG13/#8, PG13/#11, or PG13/#12 cells contained retrovirus, and the supernatants were collected for genetic modification of T cells.
The peripheral blood mononuclear cells (PBMCs) were separated using centrifugation by adding the whole blood obtained from a healthy human donor into Ficoll Paque (GE Healthcare). The separated PBMCs were cultured by adding an anti-CD3 monoclonal antibody (eBioscience) at a concentration of 100 ng/mL under the condition of human IL-2 (Novartis) at a concentration of 100 IU/mL to activate the T cell fraction (BL Levine, Cancer Gene Therapy, 2015, 22:79-84). Two to three days after the cultivation, most of the cells were T cells and included natural killer cells at a Date Recue/Date Received 2021-03-05 13 percentage of 0% to 2%. Two to three days after the activation step, the T
cells were subjected to transduction two times over two days using the retroviral supernatant and washed, and then proliferated for four to seven days in a flask. The cells were cultured in a stirring platform device (a WAVE bio-reactor system) for 12 to 28 days.
IL-2 was maintained at a concentration of 100 IU/mL. The T cells modified as such were used for an analysis experiment.
Experimental Example 1: Checking of growth rate and viability of CAR-T cells transformed with novel chimeric antigen receptors Experimental Results For the T cells prepared in Example 2, the number of cells was counted to confirm the growth rate and viability of CAR-T, and the results are shown in FIG. 8.
The number of cells and growth rate of all of the groups (#1, #2, #5, #6, #7, #8, #11, or #12) were shown to be higher than those of the control group (i.e., YYB103) depending on the day of the week, the cells were shown to grow rapidly from Day 12 of culture, and the viability was also shown to be 90% or higher (see FIG. 8).
Experimental Example 2: Checking of CAR expression rate on surface of CAR-T
cells transformed with novel chimeric antigen receptors Experimental Method (flow cytometric analysis) For flow cytometry (>30,000 events), a BD LSRII device (Becton Dickinson) and BD FACSDiva software (Becton Dickinson) were used. Specifically, the cells were washed once with PBS containing 2% bovine serum albumin before adding a PE-conjugated anti-human IL-13 monoclonal antibody (BD Pharmingen) thereto. After washing, the cells were reacted with each antibody at 4 C for 30 minutes in a state where light was blocked, and washed once, and thereafter, the expression rate of CAR
on the surface of transduced T cells was checked.
Experimental Results In order to confirm whether the 8 kinds of IL13Ra2-specific, CAR-containing polypeptides prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12) were expressed on the T cell surface, T cell culture was performed for 28 days according to Example 2, and then flow cytometric analysis was performed according to the experimental method.
As a result of the analysis, as shown in FIG. 9, the expression rate of the chimeric antigen receptors expressed on the cell surface of live T cells was shown to be in the range of 24.5% to 84.2%, and the expression of IL13Ra2-specific chimeric antigen receptors was stably maintained for 4 weeks, without additional activation or transduction of T cells (see FIG. 9).
Experimental Example 3: Checking of chimeric switch receptor expression rates of TGF-3R2 and IL-18R onthe cell surface of CAR-T cells transformed with novel chimeric antigen receptors Experimental Methods (flow cytometric analysis) For flow cytometry (>30,000 events), a BD LSRII device (Becton Dickinson) and BD FACSDiva software (Becton Dickinson) were used. Specifically, the cells were washed once with PBS containing 2% bovine serum albumin before adding a human TGF-8R2 fluorescein-conjugated antibody (FAB2411F) (BD Pharmingen) thereto.
After Date Recue/Date Received 2021-03-05 14 washing, the cells were reacted with each antibody at 4 C for 30 minutes in a state where light was blocked and washed once, and thereafter, the chimeric switch receptor expression rates of TGF-8R2 and IL-18R on the cell surface of transduced T
cells were checked.
Experimental Results In order to confirm whether the chimeric switch receptors of TGF8R2 and IL-18R
were expressed on the cell surface of CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12), T cell cultivation was performed for 14 days according to Example 2, and then flow cytometric analysis was performed according to the experimental method. As a result of the analysis, the expression rates of the chimeric switch receptors of TGF8R2 and IL-18R expressed on the cell surface of live T
cells were shown to be about 85% (see FIG. 10).
Experimental Example 4: Checking of phenotypes on the cell surface of CAR-T
cells transformed with novel chimeric antigen receptors Experimental Methods (flow cytometric analysis) For flow cytometry (>30,000 events), a BD LSRII device (Becton Dickinson) and a BD FACSDiva software (Becton Dickinson) were used. Specifically, the cells were washed once with PBS containing 2% bovine serum albumin before adding an FITC-conjugated CD45RA Ab (HI100) (Biolegend), a PE-conjugated CCR7Ab (G043H7) (Biolegend), and a PE-Cy7-conjugated CD62L Ab (DREG-56) (Biolegend) thereto.
After washing, the cells were reacted with each antibody at 4 C for 30 minutes in a state where light was blocked and washed once, and thereafter, the content of less-differentiated memory CAR-T cells and CCR7+CD45RA+CD62L+ phenotypes on the surface of transduced T cells were checked.
Experimental Results In order to confirm the content of the less-differentiated memory CAR-T cells on the cell surface of CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12), T cell cultivation was performed for 10 days according to Example 2, and then flow cytometric analysis was performed according to the experimental method.
As a result of the analysis, the content of less-differentiated memory CAR-T cells showing CCR7+CD45RA+CD62L+ phenotypes, which were expressed on live T cells, compared to a non-transduced sample (i.e., a control group) by 5% or higher, and compared to YYB103 (i.e., the transduced sample, CAR-T control group) by 10% or higher, on the surface of all of the groups of CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12), was confirmed (see FIG. 11).
Experimental Example 5: Checking of cytotoxicity of CAR-T cells transformed with novel chimeric antigen receptors against glioma cell line in which IL13Ra2 is overexpressed Experimental Methods In order to measure the cytotoxicity of IL13Ra2-specific CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12), cytotoxicity assay was performed using an LDH (Promega) kit. Specifically, CAR-T cells (effector cells) were used 10 days after activation of the cells with an anti-CD3 Ab, and the CAR-T cells (having a CAR
expression rate of 20% to 40%) were added to a 6-well plate at a ratio 1:2, in which Date Recue/Date Received 2021-03-05 15 effector: target was 1x106 cells : 2x106 cells and reacted at 37 C for 24 hours. As the used target cells, U87 cells (expressing IL13Ra2) and 293FT cells (the control group for normal cells) were used.
Experimental Results An analysis was performed to examine whether IL13Ra2-specific CAR-T cells prepared in the present invention can effectively kill the target cancer cells (U87). The method used was such that where target cancer cells (U87) and normal cells (293FT) described above were cultured together with each of the activated CAR-T cells and cytotoxicity was compared and analyzed. As shown in FIG. 12, all of the CAR-T
cells prepared according to the present invention showed a higher effect by 50% to 70% of inducing the death of the target cancer cell (U87) compared to non-transduced activated T cells. In particular, CAR-T #5, #6, and #11 of the present invention showed high cytotoxicity. In addition, CAR-T cells (#1, #2, #5, and #6) containing IL-7Ra exhibited higher overall cytotoxicity compared to CAR-T cells (#7, #8, #11, and #12) containing IL-21R8.
From the experimental result where 293FT cells, which do not express IL13Ra2, were used as the control group for normal cells, as an object to be compared with the target cells, it was confirmed that the CAR-T cells specific to IL13Ra2 showed very weak cytotoxicity (2% to 4%). This result shows that the chimeric antigen receptors used in this experiment bind specifically to IL13Ra2.
Through this Experimental Example, it was found that IL13Ra2-specific chimeric antigen receptor T cells do not show cytotoxicity to a normal cell (293FT) and significantly kill target cancer cells (U87), which express IL13Ra2.
FIG. 13 shows the results in which the cytotoxicity of the CAR-T cells, which were transformed with the chimeric antigen receptor (CAR) of the present invention, was confirmed through LDH assay after 24 hours or 96 hours without co-culture with target cells. Since the spontaneous toxicity of YYB103 is higher than that of other groups, the 8 groups are expected to show higher cytotoxicity compared to YYB103 in the co-culture with U87 cells, and the CAR-T cells (#1, #2, #5, #6, #7, #8, #11, or #12), which were transformed with the chimeric antigen receptor (CAR) of the present invention, are expected to have a comparative advantage in terms of safety and stability.
Experimental Example 6: Checking of changes in CAR expression rate when CAR-T cells, which were transformed with novel chimeric antigen receptors, are co-cultured with gliorna cell line in which IL13Ra2 is overexpressed Experimental Methods In order to check the changes in CAR expression rate when the CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12) are co-cultured with glioma cell line in which IL13Ra2 is overexpressed, the CAR-T cells (effector cells) having a CAR expression rate of 20% to 40% were used 10 days after activation of the cells with an anti-CD3 Ab. The CAR-T cells were added to a 6-well plate at a ratio 1:2, in which effector: target was 1x106 cells: 2x106 cells and reacted at 37 C for 24, 48, and 96 hours. To the sample, which was reacted for 96 hours, were added 2x106 cells of the target cells after 48 hours, and a BD LSRII device (Becton Dickinson) and BD
FACSDiva software (Becton Dickinson) were used for flow cytometry (>30,000 events).
Date Recue/Date Received 2021-03-05 16 Specifically, the cells were washed once with PBS containing 2% bovine serum albumin before adding a PE-conjugated anti-human IL-13 monoclonal antibody (BD
Pharmingen) thereto. After washing, the cells were reacted with each antibody at 4 C for 30 minutes in a state where light was blocked and washed once, and thereafter, the changes in the CAR expression rate were checked.
Experimental Results In order to confirm the changes in the CAR expression rate when the 8 kinds of IL13Ra2-specific CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12) are co-cultured with glioma cell line in which IL13Ra2 is overexpressed, the T cells were cultured for 10 days, and then flow cytometric analysis was performed according to the experimental method according to Example 2. As a result of the analysis, as shown in FIG. 14, the CAR-T cells which were co-cultured with U87 cells for 24 hours showed a smaller change in CAR expression in all of the 8 groups compared to that of YYB103 (FIG. 14).
In co-culture with U87 cells for 48 hours, the change in CAR expression was shown to be smaller than that of YYB103 in all of the 8 groups (FIG. 15).
In co-culture with U87 cells for 96 hours, the change in CAR expression was shown to be smaller than that of YYB103 in 5 groups (i.e., #1, #2, #5, #6, and #7) (FIG. 16). The CAR expression of YYB103 after co-culture with U87 cells for 96 hours was shown to be insignificant (FIG. 16).
The changes in CAR expression during the co-culture with glioma cell line, in which IL13Ra2 is overexpressed, the changes in the CAR expression rate without co-culture with target cells or during the co-culture with 293FT cells (which were used as the control group for normal cells) can confirm that the chimeric antigen receptor (CAR) according to the present invention is expected to show excellent in vivo persistence and anti-tumor effects of CAR-T cells in a hostile tumor microenvironment, while simultaneously reducing toxicity.
Experimental Example 7: Checkind of chandes in chimeric switch receptor expression rate of TGFOR2 and IL-18R when CAR-T cells which were transformed with novel chimeric antiden receptors are co-cultured with dlioma cell line, in which IL13Ra2 is overexpressed Experimental Methods In order to check the changes in the chimeric switch receptor expression rate of TGF3R2 and IL-18R when CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12) are co-cultured with glioma cell line, in which IL13Ra2 is overexpressed, the CAR-T cells (effector cells) having a CAR expression rate of 20% to 40%
were used days after activation of the cells with an anti-CD3 Ab. The CAR-T cells were added to a 6-well plate at a ratio 1:2, in which effector: target was 1x106 cells:
2x106 cells and reacted at 37 C for 24, 48, and 96 hours. To the sample, which was reacted for hours, were added 2x106 cells of the target cells after 48 hours, and a BD
LSRII device (Becton Dickinson) and BD FACSDiva software (Becton Dickinson) were used for flow cytometry (>30,000 events). Specifically, the cells were washed once with PBS
containing 2% bovine serum albumin before adding an anti-human TGF-31R2 fluorescein-conjugated antibody (FAB2411F) (BD Pharmingen) thereto. After washing, Date Recue/Date Received 2021-03-05 17 the cells were reacted with each antibody at 4 C for 30 minutes in a state where light was blocked and washed once, and thereafter, the changes in the chimeric switch receptor expression rate of TGF8R2 and IL-18R were checked.
Experimental Results In order to confirm the changes in the chimeric switch receptor expression rate of TGF8R2 and IL-18R when the 8 kinds of IL13Ra2-specific CAR prepared in Example
[Invention Title]
CHIMERIC ANTIGEN RECEPTOR FOR SOLID CANCER AND T CELLS
EXPRESSING CHIMERIC ANTIGEN RECEPTOR
[Technical Field]
The present invention relates to a novel chimeric antigen receptor (CAR) for effectively treating blood cancer or solid cancer, and to a CAR-T cell in which a chimeric antigen receptor targeting a specific cancer antigen is expressed. In addition, the present invention relates to a vector which expresses a novel CAR for effectively treating blood cancer or solid cancer.
More specifically, the present invention relates to a chimeric antigen receptor comprising an antigen binding domain; a hinge region; a transmembrane domain;
a costimulatory domain; and a cytoplasmic signaling domain, in which any one immunoreceptor tyrosine-based activation motif (hereinafter described as "ITAM") present within a CD3 domain, which is a cytoplasmic signaling domain, is mutated for excellent in vivo persistence and anti-tumor effects of CAR-T cells, and to a CAR-T cell in which the chimeric antigen receptor is expressed.
Further, in order to induce a cytokine signal in an antigen-dependent manner during stimulation of cancer antigens, a cleaved cytoplasmic domain of interleukin-7 receptor-a (IL7Ra) or interleukin-2 receptor-p (IL-2R[3) in addition to the CD3 signaling domain and a 4-1BB costimulatory domain was added to a CAR gene so as to reduce the differentiation and exhaustion of CAR-T cells caused by CAR tonic signaling of CAR-T cells, thereby making it capable of exhibiting excellent in vivo persistence and anti-tumor effects.
In addition, in the present invention, a chimeric switch receptor was introduced to convert the immunosuppressive signal in the hostile tumor microenvironment into an activation signal in CAR-T cells.
In addition, in the present invention, the cytokine IL-21 is released out of CAR-T
cells so as to aid the activation of innate immune-related cells and increase the content of less-differentiated memory CAR-T cells.
The chimeric antigen receptor according to the present invention can show excellent in vivo persistence and anti-tumor effects of CAR-T cells in a hostile tumor microenvironment, and can simultaneously reduce toxicity, thereby reducing side effects in the patient while improving the treatment effect. In addition, it is possible to expand the scope of use of the present invention by preparing a chimeric antigen receptor-provided allogenic (non-self-derived) CAR-T treatment according to the present invention.
[Background]
T cells expressing chimeric antigen receptor (CAR) (CAR-T cells) refers to a type of T cell with a recombinant gene, in which a gene encoding a receptor that recognizes cancer cell surface antigens specifically expressed on the surface of cancer cells is Date Recue/Date Received 2021-03-05 1 introduced into a T cell to kill cancer cells.
Dr. Zelig Eshhar, an Israeli chemist and immunologist at the Weizmann Institute of Science, et al. previously proposed a hypothesis that when T cells are artificially made with a receptor that binds to an antigen specifically expressed in a cancer cell, it is possible to target only cancer cells and trigger an immune response to kill cancer cells. They succeeded in preparing T cells provided with a chimeric antigen receptor, and this was reported in PNAS in 1989.
In the CAR-T cells prepared in the early days (i.e., the first-generation CAR-T
cells), only CD3 was used as a signaling domain. However, these CAR-T cells had drawbacks in that the treatment effect was insignificant and that the persistence was also short. Therefore, efforts have been made to improve the reactivity of CAR-T cells, and as a result, second-generation CAR-T cells were prepared in which a costimulatory domain (0D28 or 0D137/4-1BB) and CD3 were combined, and these CAR-T cells showed an increase in the number of CAR-T cells remaining in the body compared to the first-generation CAR-T cells.
In the second-generation CAR-T cells, only one kind of costimulatory domain was used, and CAR-T cells using two kinds of costimulatory domains are referred to as third-generation CAR-T.
With regard to the method of treating cancer using CAR-T cells, three late-stage chronic lymphoid leukemia (CCL) patients were injected with cytotoxic T cells which were modified to recognize CD19. As a result, leukemia was completely cured in two of the patients, and there were reports that the conditions lasted for about 10 months (N
Engl J Med 2011, 365:725-733, August 25, 2011; Sic Transl Med 2011 Aug 10, 3(95):95ra73). In particular, the CAR-T used corresponded to the second-generation CAR-T cells, in which 4-1BB was used as the costimulatory domain and CD3 was used as the signaling domain. The antigen binding domain of the CAR-T cells recognizes CD19 found on the surface of leukemia cancer cells as an antigen.
In addition, there was a report that when CTL019 was administered to patients with acute leukemia, 27 out of 30 patients experienced complete remission, 67%
of the patients experienced complete remission for over 2 years, and 78% of the patients survived for two years. Considering that the target patients above were relapsed or refractory patients, these results were very surprising (N Engl J Med 2014, 371:1507-1517, October 16, 2014).
Currently, clinical trials for various blood cancers (e.g., lymphoma, myeloma, etc.) are in progress with regard to therapeutic methods using various CAR-T cells, and CAR-T, as a pharmaceutical drug that can be used for the treatment of blood cancer, has entered the market.
However, there are several obstacles to using CAR-T cells for the treatment of solid cancer. For example, when CAR-T cells are administered intravenously to a cancer patient, it is difficult for CAR-T cells to migrate to the tumor site;
and in a hostile tumor microenvironment, CAR-T cells are functionally inhibited while simultaneously exhibiting limited persistence and proliferation.
Therefore, there is a need to develop CAR-T cells which can treat solid cancer by overcoming these problems.
Date Recue/Date Received 2021-03-05 2 [Detailed Description of the Invention]
[Technical Problem]
An object of the present invention is to provide CAR platforms, CAR-T cells, and CAR expression vectors which have remarkably excellent therapeutic effects in solid cancer compared to the previously known CAR-T cells.
The CAR-T cell therapeutic of the present invention for treating solid cancer comprises: (1) a mutated ITAM introduced into the cytoplasmic signaling domain for excellent in vivo persistence and antitumor effects of CAR-T cells; and/or (2) CAR tonic signaling controlled by adding a cleaved cytoplasmic domain of IL7Ra or IL-2R[3 in addition to a CD3 signaling domain and a 4-1BB costimulatory domain to a CAR
gene so as to induce a cytokine signal in an antigen-dependent manner; and/or (3) a chimeric switch receptor introduced so as to convert the immunosuppressive signal in the hostile tumor microenvironment into an activation signal in CAR-T cells; and/or (4) cytokine IL-21 allowed to release out of CAR-T cells so as to aid the activation of innate immune-related cells and increase the content of less-differentiated memory CAR-T
cells.
Due to CAR tonic signaling, CAR-T cells promote anergy, differentiation, exhaustion, and activation-induced CAR-T cell death. In order to increase the therapeutic efficacy of CAR-T cells for treating solid cancer, excellent in vivo persistence and anti-tumor effects of CAR-T cells are essential. Therefore, it is necessary to control the CAR tonic signaling system in strategies for solid cancer treatment.
Since the CAR tonic signaling can be controlled by substitution of a cytosolic signaling domain of CAR-T cell, an antigen-dependent cytokine signal was added into CAR-T cell. Additionally, in order to control the CAR tonic signaling that may occur due to uncontrolled excessive ITAM-based signaling within the CD3 domain, one mutated ITAM was introduced into the cytoplasmic signaling domain. By controlling CAR
tonic signaling, the excellent in vivo persistence and anti-tumor effects of CAR-T
cells were made to last longer.
In addition, a chimeric switch receptor was introduced, which converts the inhibitory signal of TGF-r3 (i.e., an immunosuppressive cytokine overexpressed in a hostile tumor microenvironment) into an activation signal in CAR-T cells. In addition, it was prepared such that cytokine IL-21 could be released out of CAR-T cells so as to aid the activation of innate immune-related cells and increase the content of less-differentiated memory CAR-T cells.
The chimeric antigen receptor of the present invention can be used to prepare CAR-T cell therapeutics for various carcinomas by changing the cancer antigen target in the antigen binding domain.
[Technical Solution]
To solve the problems described above, the present invention provides a polypeptide comprising a chimeric antigen receptor, which comprises an antigen binding domain; a hinge region; a transmembrane domain; a costimulatory domain; and a signaling domain. In particular, the costimulatory domain may comprise a 4-1BB
Date Recue/Date Received 2021-03-05 3 domain or 0D28 domain or both a 4-1BB domain and a 0D28 domain, in which the signaling domain comprises a CD3 domain.
In the present invention, the polypeptide comprises IL-7Ra or a part thereof interposed between the 4-1BB domain and the CD3 domain. In particular, a part of IL-7Ra may be a sequence of SEQ ID NO: 15, and a part of IL-21R8 may be a sequence of SEQ ID NO: 17. A part of the cytoplasmic domain of the IL-7Ra (SEQ ID NO: 15) is a sequence between the 266th and the 328th positions in the entire sequence of IL-7Ra of SEQ ID NO: 14, and a part of the cytoplasmic domain of the IL-21R8 (SEQ ID NO:
17) is a sequence between the 266th and the 369th positions in the entire sequence of of SEQ ID NO: 16.
For example, in vivo persistence and anti-tumor effects of CAR-T cells were increased by controlling the CAR tonic signaling by linking a cleaved cytoplasmic domain of IL-7Ra (amino acids at positions from 265 to 328; SEQ ID NO: 15) or cleaved cytoplasmic domain of IL2R8 (amino acids at positions from 266 to 369; SEQ ID
NO: 17) between 4-1BB (SEQ ID NO: 11) as a costimulatory domain and mutant CD3 (in which the positions from 91 to 113 of SEQ ID NO: 18 are substituted with either SEQ
ID
NO: 31 or SEQ ID NO: 32), in a CAR which is specific to IL-13Ra2, based on the known anti-IL13Ra2 CAR-T cells (YYB-103 of SEQ ID NO: 22: see PCT International Publication No. WO 2017/023138).
In the present invention, three immunoreceptor tyrosine-based activation motifs (ITAMs) are included within the CD3 domain, and in a third ITAM region among these, it is preferred that a first motif, YxxL, and a second motif, YxxL, be substituted (in which x and y each represent any amino acid) (see FIG. 3).
Generally, the ITAM within the CD3 domain includes two forms of YxxL (or isoleucine (I)) containing tyrosine (Y), which is spaced apart from leucine (L) or isoleucine (I) by a distance of a two amino acid sequence. Since the YxxL/I
forms are usually present to be spaced apart by 6 to 8 amino acids, the ITAM can be represented by the structure of YxxL/Ix(6_8)Y)o(L/1.
In the present invention, three ITAMs (positions 21 to 35; positions 60 to 75;
and positions 91 to 105 of SEQ ID NO: 18) are included within the CD3 domain, which is represented by SEQ ID NO: 18, in which in the CD3 domain, it is preferred that in a third ITAM region, a first motif, YxxL (positions 91 to 94 of SEQ ID NO: 18), be substituted with YxxQ, and a second motif region, YxxLHM (positions 102 to 107 of SEQ ID NO: 18), be substituted with YxYVTM, or in a third ITAM region, a first motif, YxxL (positions 91 to 94 of SEQ ID NO: 18), be substituted with YyyL, and a second motif, YxxL (positions 102 to 105 of SEQ ID NO: 18), be substituted with YxxQ
(in which x and y each represent any amino acid).
For example, in the present invention, the region including a third ITAM
region of the CD3 domain may be mutated to a sequence of SEQ ID NO: 31 or 32.
Specifically, a chimeric antigen receptor (CAR) can be used, in which among the three ITAM motifs present within the CD3 domain (see SEQ ID NO: 18) (i.e., a known signaling domain of anti-IL13Ra2 CAR-T cells (YYB-103)), the sequence of a third ITAM
motif (i.e., positions 91 to 106 of SEQ ID NO: 18) is mutated from "YQGLSTATKDTYDALHMQALPPR" to "YLPQSTATKDTYDYVTMQALPPR" (SEQ ID
Date Recue/Date Received 2021-03-05 4 NO: 31); or among the three ITAM motifs present within the CD3 domain (see SEQ
ID
NO: 18) (i.e., a known signaling domain of anti-IL13Ra2 CAR-T cells (YYB-103)), the sequence of a third ITAM motif (i.e., positions 91 to 105 of SEQ ID NO: 18) is mutated from "YQGLSTATKDTYDALHMQALPPR" to "YLSLSTATKDTYLPQHMQALPPR" (SEQ
ID NO: 32) (see FIG. 3).
In particular, in the present invention, it is preferred that in a third ITAM
region of the CD3 domain, a first motif, YxxL (positions 91 to 94 of SEQ ID NO: 18), be substituted with YxxQ, and a second motif, YxxLHM (positions 102 to 106 of SEQ
ID
NO: 18), be substituted with YxYVTM, or in a third ITAM region of the CD3 domain, a first motif, YxxL (positions 91 to 94 of SEQ ID NO: 18), be substituted with YyyL, and a second motif, YxxL (positions 102 to 105 of SEQ ID NO: 18), be substituted with YxxQ
(in which x and y each represent any amino acid).
The polypeptide of the present invention may further contain a cytokine. For example, the polypeptide of the present invention may contain IL-21 as a cytokine to be added. In particular, it is preferred that the cytokine be linked to a chimeric antigen receptor by a self-cleaving peptide.
As the self-cleaving peptide, for example, the known 2A peptides of SEQ ID
NOS: 40 to 43 may be used. The self-cleaving peptide undergoes a post-translational cleavage, in which the peptide bond between a proline (P) and a glycine (G) in the C-terminus is digested by cleavage. Therefore, the proteins located upstream and downstream of the self-cleaving peptide are expressed independently of each other.
Accordingly, when the polypeptide according to the present invention is expressed in a T cell, the cytokine can be separated from a chimeric antigen receptor (CAR), and the separated cytokine can be released to the outside of the T
cell. Innate immune-related cells can be activated by the separated cytokine (e.g., IL-21), and thereby the content of less-differentiated CAR-T cells can be increased (see FIG. 5).
That is, in vitro differentiation is prevented due to the IL-21 expression during the process of CAR-T cell preparation, which results in an increase of the content of less-differentiated memory CAR-T cells, thereby making it possible to provide excellent in vivo persistence and enhanced anti-tumor effects of CAR-T cells, and which additionally results in activation of the innate immune cells around the solid cancer, thereby making it possible to more effectively treat solid cancer.
The polypeptide of the present invention may further include a TGF-8R2 exodomain, an IL18R transmembrane domain, and an IL18R endodomain, in addition to a chimeric antigen receptor. In particular, it is preferred that an IL18R
transmembrane domain be included between the TGF-8R2 exodomain and the IL18R endodomain (see FIG. 4).
In the present invention, it is preferred that the TGF-8R2 exodomain be linked to a chimeric antigen receptor by a self-cleaving peptide. Therefore, when the polypeptide according to the present invention is expressed in T cells, the polypeptide comprising a TGF-8R2 exodomain, an IL18R transmembrane domain, and an IL18R endodomain is separated from a chimeric antigen receptor (CAR). The TGF-8R2 domain of the separated polypeptide is exposed to the outside of T cells, the IL18R
transmembrane domain is located in the cell membrane of T cells, and the IL18R endodomain is located Date Recue/Date Received 2021-03-05 5 in the cytoplasm of T cells.
Accordingly, when TGF-8, which is an immunosuppressive cytokine, is present outside of T cells, TGF-8 binds to the TGF-8R2 exodomain, thereby activating the IL18R endodomain. That is, the inhibitory signal of TGF-8 (i.e., an immunosuppressive cytokine in a hostile tumor microenvironment) is converted into an activation signal of the cytokine IL-18 in CAR-T cells. Therefore, a polypeptide, which includes the TGF-8R2 exodomain, IL18R transmembrane domain, and IL18R endodomain, becomes a chimeric switch receptor.
IL-18, which is a cytokine, inhibits expression of immunosuppressive materials in T cells and differentiation of regulatory T cells, and enhances immune responses against cancer cells. When the effects of the CAR-T cell therapeutic by IL-18 in advanced solid tumors were examined, it was confirmed that Fox01 expression in CD4+
T cells was decreased by IL-18, and additionally that T-bet expression was increased by IL-18 in CD8+ T cells.
Ultimately, IL-18 shows an excellent anticancer effect in advanced solid cancer through the T-bethigh Fox01low CAR-T, persistent CAR-T cells. In particular, in the present invention, a chimeric switch receptor, which includes a TGF-8R2 exodomain (amino acid positions of 23 to 166; SEQ ID NO: 19), which is a receptor for binding to TGF-8 (i.e., an immunosuppressive cytokine around solid cancer); and a transmembrane domain and an endodomain of IL-18R (amino acid positions of 323 to 541; SEQ ID NO: 20), which is a cytokine receptor, was prepared to be expressed in CAR-T cells, thereby changing the immunosuppressive signal into an activation signal in CAR-T cells by making a reverse use of the hostile tumor microenvironment.
As described above, even a polypeptide, which additionally includes a TGF-8R2 exodomain, an IL18R transmembrane domain, and an IL18R endodomain in the chimeric antigen receptor, may further include the cytokine described above (e.g., IL-21).
That is, the cytokine described above (e.g., IL-21) may be linked by a self-cleaving peptide to the IL18R endodomain of the polypeptide, which additionally includes a TGF-8R2 exodomain, an IL18R transmembrane domain, and an IL18R
cytoplasmic endodomain in the chimeric antigen receptor.
Accordingly, when the polypeptide according to the present invention is expressed in T cells, the polypeptide which includes the TGF-8R2 exodomain, transmembrane domain, and IL18R endodomain can be separated from the chimeric antigen receptor (CAR), and additionally, the cytokine can also be independently separated. Therefore, the separated cytokine can be released to the outside of T cells, the TGF-8R2 exodomain is exposed to the outside of the T cells, the IL18R
transmembrane domain is located in the cell membrane of T cells, and the IL18R
endodomain is located in the cytoplasm of T cells.
The polypeptide according to the present invention may be, for example, a polypeptide represented by any one of SEQ ID NOS: 24 to 30 and 34 to 37.
In addition, the present invention relates to a CAR-T cell in which the CAR-containing polypeptide is expressed as described above.
In addition, the present invention relates to a chimeric antigen receptor (CAR) expression vector, which includes an antigen binding domain; a hinge region; a Date Recue/Date Received 2021-03-05 6 transmembrane domain; a costimulatory domain; and a cytoplasmic signaling domain.
In particular, the CAR expression vector includes a nucleic acid encoding a CAR, in which the nucleic acid encoding a CAR comprises a nucleic acid encoding a 4-domain as the costimulatory domain and a nucleic acid encoding a CD3 domain as the signaling domain, and further comprises a nucleic acid encoding IL-7Ra or a part thereof or a nucleic acid encoding IL-2Rp or a part thereof between the nucleic acid encoding the 4-1BB domain and the nucleic acid encoding the CD3 domain. In particular, a part of IL-7Ra may be, for example, a sequence of SEQ ID NO: 15, and a part of IL-2Rp may be, for example, a sequence of SEQ ID NO: 17.
In the present invention, the nucleic acid encoding the CD3 domain may be a nucleic acid encoding a CD3 domain, in which in a third ITAM region among the three ITAMs present within the CD3 domain, a first motif, YxxL, and a second motif, YxxLHM, are substituted. Preferably, the nucleic acid encoding the CD3 domain may be a nucleic acid encoding a CD3 domain, in which in a third ITAM region, a first motif, YxxL, is substituted with YxxQ, and a second motif region, YxxLHM, is substituted with YxYVTM, or one in which a first motif, YxxL, is substituted with YyyL, and a second motif region, YxxL, is substituted with YxxQ (in which x and y each represent any amino acid).
In addition, the CAR expression vector of the present invention may further include a nucleic acid encoding cytokine IL-21. In particular, the nucleic acid encoding cytokine IL-21 is linked to a nucleic acid encoding a CAR through a nucleic acid encoding a self-cleaving peptide.
In addition, the CAR expression vector of the present invention may further include a nucleic acid encoding a TGF-pR2 exodomain, a nucleic acid encoding an IL18R transmembrane domain, and a nucleic acid encoding an IL18R endodomain.
In particular, the nucleic acid encoding the TGF-pR2 exodomain is linked to a nucleic acid encoding a CAR through a nucleic acid encoding a self-cleaving peptide.
As described above, the CAR expression vector, which includes a nucleic acid encoding a TGF-pR2 exodomain, a nucleic acid encoding an IL18R transmembrane domain, and a nucleic acid encoding an IL18R endodomain, may further include a nucleic acid encoding cytokine IL-21. In particular, the nucleic acid encoding cytokine IL-21 is linked to a nucleic acid encoding an IL18R endodomain through a nucleic acid encoding a self-cleaving peptide.
The present invention relates to a CAR-T cell prepared by introducing the CAR
expression vector described above.
In addition, the present invention relates to an anticancer agent containing the CAR-T cell described above. The anticancer agent of the present invention containing the CAR-T cell may further contain a pharmaceutically acceptable additive as necessary.
In the CAR-containing polypeptides according to the present invention, CAR-T
cells for various carcinomas may be prepared by changing the cancer antigen target of the antigen binding domain as necessary. For example, the antigen binding domain may be prepared as those which bind to IL13Ra2, an antigen associated with an angiogenesis activity (anti-angiogenesis), and antigens such as EGFRvIll, EphA2, aVpMesothelin, and glypican1. That is, if any ligand or antibody targeting IL-13Ra2, Date Recue/Date Received 2021-03-05 7 anti-angiogenesis, EGFRvIll, EphA2, aVpglypican1, and mesothelin (see SEQ ID
NOS: 1 to 7 and 33) is introduced, these CAR-T cells may be used as an anticancer agent for these targets. Therefore, it is possible to prepare CAR-T cells having an anti-cancer effect against a specific carcinoma.
In the case of EGFRvIll (i.e., a major tumor antigen expressed in glioblastoma, lung cancer, etc.), in order to reduce side effects of CAR-T cells that appear through a non-specific binding, a CAR expression rate and persistence in CAR-T cells were optimized while simultaneously changing the target sequence so as to minimize the binding affinity with EGFR wild-type while maintaining the specificity for EGFRvIll.
While changing positions 52 to 57 of SEQ ID NO: 3 from STGGYN to DPENDE
(a CDR2 part of a heavy chain), position 101 of SEQ ID NO: 3 from S to G (a CDR3 part of a heavy chain), and position 229 of SEQ ID NO: 3 from V to G (a CDR3 part of a light chain) in the target sequence (SEQ ID NO: 33), a CAR expression rate and persistence in CAR-T cells were improved using CD8 signal sequence (SEQ ID NOS: 34 and 35).
In order to increase a CAR expression rate and persistence in CAR-T cells targeting aV[3, a Gaussia princeps luciferase signal sequence or CD8 signal sequence was used (SEQ ID NOS: 36 and 37).
[Advantageous Effects]
The CAR-T cells disclosed in the present invention have an excellent expression rate and persistence; therefore, these cells show the effects of persistence and anti-tumor effects in the human body, thereby having an improved therapeutic effect against solid cancer.
[Brief Description of Drawings]
FIG. 1 shows a diagram illustrating a process of effectively treating solid cancer by a CAR-containing polypeptide according to the present invention.
FIG. 2 shows a diagram and a table illustrating an antigen binding domain which can be used for a CAR-containing polypeptide platform according to the present invention.
FIG. 3 shows diagrams illustrating the introduction of a cytokine signaling domain into CAR-T cells so as to control CAR tonic signaling in the CAR-containing polypeptide according to the present invention.
FIG. 4 shows diagrams illustrating the introduction of a chimeric switch receptor so as to convert an immune suppressive signal in a hostile tumor microenvironment into an activation signal in CAR-T cells.
FIG. 5 shows diagrams illustrating the release of cytokine IL-21 to the outside of CAR-T cells so as to aid the activation of innate immune-related cells and to increase the content of less-differentiated memory CAR-T cells.
FIG. 6 shows diagrams illustrating the structures of CAR-containing polypeptides according to the present invention.
FIG. 7 shows diagrams illustrating the self-cleaving peptides used in the CAR-containing polypeptides according to the present invention.
FIG. 8 shows a graph and tables illustrating the growth rate and viability of CAR-Date Recue/Date Received 2021-03-05 8 T cells, which are transformed with the CAR of the present invention.
FIG. 9 shows graphs and a table illustrating the analysis results of the CAR
expression rate of CAR-T cells, which were transformed with the CAR of the present invention, by flow cytometry analysis.
FIG. 10 shows a graph and a table illustrating the analysis results of the chimeric switch receptor expression rate of TGF-8R2 and IL-18R of CAR-T cells, which were transformed with the CAR of the present invention, by flow cytometry analysis.
FIG. 11 shows the results of Day 10 phenotypes of CAR-T cells, which were transformed with the CAR of the present invention, by flow cytometry analysis.
FIG. 12 shows a graph, which illustrates the cytotoxicity of CAR-T cells, which were transformed with the CAR of the present invention, confirmed through LDH
assay after 24 hours of co-culture with U87 cells (human brain cancer cell line) and target cells (which were used as a control group for normal cells); and a table, which illustrates the purity and viability of CAR-T cells used in a CAR-T
cytotoxicity test.
FIG. 13 shows graphs illustrating the analysis results of spontaneous toxicity of CAR-T cells, which were transformed with the CAR of the present invention, after 24 hours or 96 hours.
FIGS. 14 to 16 each show graphs and a table illustrating the analysis results of changes in a CAR expression rate of CAR-T cells, which were transformed with the CAR of the present invention, after 24 hours (FIG. 14), 48 hours (FIG. 15), and 96 hours (FIG. 16) of co-culture of the CAR-T cells with U87 cells (human brain cancer cell line) and 293FT target cells (which were used as a control group for normal cells), by flow cytometry analysis.
FIG. 17 shows graphs and a table illustrating the analysis results of changes in the chimeric switch receptor expression rate of TGF-8R2 and IL-18R of CAR-T
cells, which were transformed with the CAR of the present invention, after 24 hours of co-culture of the CAR-T cells, which were transformed with the CAR of the present invention, with U87 cells (human brain cancer cell line) and 293FT target cells (which were used as a control group for normal cells), by flow cytometry analysis.
FIG. 18 shows graphs illustrating the comparison results of IFN-y cytokine production after 24 hours and 48 hours of co-culture of CAR-T cells, which were transformed with the CAR of the present invention, with U87 cells (human brain cancer cell line) and 293FT target cells (which were used as a control group for normal cells).
FIG. 19 shows graphs illustrating the comparison results of IL-21 cytokine production after 24 hours and 48 hours of co-culture of CAR-T cells, which were transformed with the CAR of the present invention, with U87 cells (human brain cancer cell line) and 293FT target cells (which were used as a control group for normal cells).
FIG. 20 shows a graph and tables illustrating the growth rate and viability of CAR-T cells, which were transformed with a chimeric antigen receptor (CAR), when the cancer antigen is EGFRvIll, in which the CAR expression rate and persistence in CAR-T cells were optimized, while simultaneously changing the target sequence so as to minimize the binding affinity for EGFR wild-type, where the specificity for EGFRvIll is maintained so as to reduce the side effects of CAR-T cells that appear through non-specific binding (YYB105, #13, #14), and a graph and tables illustrating the growth rate Date Recue/Date Received 2021-03-05 9 and viability of CAR-T cells, which were transformed with a chimeric antigen receptor (CAR), when the cancer antigen is aV[3, in which the CAR expression rate in CAR-T
cells was optimized (YYB107, #15, #16).
FIG. 21 shows a graph illustrating the CAR expression rate in CAR-T cells, which were transformed with a chimeric antigen receptor (CAR), when the cancer antigen is EGFRvIll, in which the CAR expression rate in CAR-T cells was optimized, while simultaneously changing the target sequence so as to minimize the binding affinity for EGFR wild-type, where the specificity for EGFRvIll is maintained so as to reduce the side effects of CAR-T cells that appear through non-specific binding (YYB105, #13, #14), and a graph illustrating the CAR expression rate in CAR-T cells, which were transformed with a chimeric antigen receptor (CAR), when the cancer antigen is aV[3, in which the CAR expression rate in CAR-T cells was optimized (YYB107, #15, #16).
[Best Mode for Carrying Out the Invention]
Hereinafter, the present invention will be described in detail through examples.
However, it should be noted that the following Examples are only for illustrating the present invention, and the spirit and technical scope of the present invention are not limited in any sense. Further, it should be understood that the present invention is not limited to the precise arrangement and means of the embodiments shown in the drawings, which are cited as reference.
Example 1: Preparation of novel chimeric antigen receptor (CAR) platform for effective treatment by specifically binding to IL13Ra2 overexpressed in solid cancer cells With regard to human IL13 (P35225.1), human CD3 (P20963-1), human CD8A
(P01732), human 0D28 (P10747), human CD3 (P20963), human 41BB (Q07011), IL7RA (P16871), IL2RB (P14784), TGFR2 (P37173), IL18R (Q13478), IL21 (Q9HBE4), IL2 (P60568), T2A, P2A, and human kappa light chain signal sequence (HuVHCAMP), CAR-containing polypeptides were prepared using a chimeric antigen-containing polypeptide (see SEQ ID NOS: 23 to 30 and 34 to 37; and #1, #2, #5, #6, #7, #8, #11, and #12 of FIG. 6) expression vector consisting of codon-optimized synthetic DNA, using scientific literature and publicly available databases (see FIGS. 6 and 7).
Specifically, T cells, in which a CAR-containing polypeptide is expressed, can be prepared by preparing a vector that expresses a CAR-containing polypeptide via conjugation of synthetic DNA to an MFG retrovirus expression vector digested with Xhol/Notl, followed by transduction of the vector into the T cells.
The completed structure of polypeptide #1 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human IL13.E11K.R64D.S67D.R107K mature protein sequence (see YYB-103 of PCT
International Publication No. WO 2017/023138), three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein (see YYB-103 of PCT International Publication No. WO 2017/023138), a hinge region of human CD8a, a human CD8 transmembrane domain, a costimulatory signal domain of Date Recue/Date Received 2021-03-05 10 cytoplasmic 4-1BB, a cleaved cytoplasmic domain of cytokine IL-7RA (amino acid positions of 265 to 328; SEQ ID NO: 15), a mutant CD3 (positions 91 to 113 of SEQ ID
NO: 18 are mutated to a sequence of SEQ ID NO: 31), T2A, a CD8A signal sequence (leader sequence), a chimeric switch receptor which comprises TGF-8R2 exodomain (amino acid positions of 23 to 166; SEQ ID NO: 19) and a transmembrane domain and an endodomain of IL-18R (i.e., a cytokine receptor) (amino acid positions of 323 to 541;
SEQ ID NO: 20), P2A, a human 1L2 signal sequence (leader sequence), an IL-21 (amino acid positions of 23 to 155; SEQ ID NO: 21) sequence, and an Xhol/Notl cleavage site (see CAR-containing polypeptide #1 of FIG. 6; and FIG. 7).
The completed structure of CAR-containing polypeptide #2 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human 11_13.E11K.R64D.S67D.R107K mature protein sequence, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8a, a human CD8 transmembrane domain, a costimulatory signal domain of cytoplasmic 4-1BB, a cleaved cytoplasmic domain of a cytokine IL-7RA (amino acid positions of 265 to 328; SEQ ID NO: 15), a mutant CD3 (positions 91 to 113 of SEQ ID NO: 18 are mutated to a sequence of SEQ ID NO: 31), T2A, a CD8A signal sequence (leader sequence), a chimeric switch receptor which comprises TGF-8R2 exodomain (amino acid positions of 23 to 166; SEQ ID NO: 19) and a transmembrane domain and an endodomain of IL-18R (i.e., a cytokine receptor) (amino acid positions of 323 to 541;
SEQ ID NO: 20), and an Xhol/Notl cleavage site (see CAR-containing polypeptide #2 of FIG. 6; and FIG. 7).
The completed structure of CAR-containing polypeptide #5 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human 11_13.E11K.R64D.S67D.R107K mature protein sequence, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8a, a human CD8 transmembrane domain, a costimulatory signal domain of cytoplasmic 4-1BB, a cleaved cytoplasmic domain of a cytokine IL-7RA (amino acid positions of 265 to 328; SEQ ID NO: 15), a mutant CD3 (positions 91 to 113 of SEQ ID NO: 18 are mutated to a sequence of SEQ ID NO: 31), T2A, a human 1L2 signal sequence, an (amino acid positions of 23 to 155; SEQ ID NO: 21) sequence, and an Xhol/Notl cleavage site (see CAR-containing polypeptide #5 of FIG. 6; and FIG. 7).
The completed structure of CAR-containing polypeptide #6 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human 11_13.E11K.R64D.S67D.R107K mature protein sequence, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8a, a human CD8 transmembrane domain, a costimulatory signal domain of cytoplasmic 4-1BB, a cytoplasmic domain of a cytokine IL-7RA (amino acid positions of 265 to 328;
Date Recue/Date Received 2021-03-05 11 SEQ ID NO: 15), a mutant CD3 (positions 91 to 113 of SEQ ID NO: 18 are mutated to a sequence of SEQ ID NO: 31), and an Xhol/Notl cleavage site (see CAR-containing polypeptide #6 of FIG. 6; and FIG. 7).
The completed structure of polypeptide CAR-containing #7 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human 11_13.E11K.R64D.S67D.R107K mature protein sequence, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8a, a human CD8 transmembrane domain, a costimulatory signal domain of cytoplasmic 4-1BB, a cleaved cytoplasmic domain of a cytokine IL2R8 (amino acid positions of 266 to 369; SEQ ID NO: 17), a mutant CD3 (positions 91 to 113 of SEQ ID NO: 18 are mutated to a sequence of SEQ ID NO: 32), T2A, a CD8A signal sequence, a chimeric switch receptor which comprises a TGF-8R2 exodomain (amino acid positions of 23 to 166; SEQ ID NO: 19) and a transmembrane domain and an endodomain of IL-18R
(i.e., a cytokine receptor) (amino acid positions of 323 to 541; SEQ ID NO: 20), P2A, a human 1L2 signal sequence (leader sequence), an IL-21 (amino acid positions of 23 to 155; SEQ ID NO: 21) sequence, and an Xhol/Notl cleavage site (see #7 of FIG.
6; and FIG. 7).
The completed structure of CAR-containing polypeptide #8 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human 11_13.E11K.R64D.S67D.R107K mature protein sequence, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8a, a human CD8 transmembrane domain, a costimulatory signal domain of cytoplasmic 4-1BB, a cleaved cytoplasmic domain of a cytokine IL2R8 (amino acid positions of 266 to 369; SEQ ID NO: 19), a mutant CD3 (positions 91 to 113 of SEQ ID NO: 18 are mutated to a sequence of SEQ ID NO: 32), T2A, a CD8A signal sequence, a chimeric switch receptor which comprises a TGF-8R2 exodomain (amino acid positions of 23 to 166; SEQ ID NO: 19) and a transmembrane domain and an endodomain of IL-18R
(i.e., a cytokine receptor) (amino acid positions of 323 to 541; SEQ ID NO: 20), and an Xhol/Notl cleavage site (see CAR-containing polypeptide #8 of FIG. 6; and FIG.
7).
The completed structure of CAR-containing polypeptide #11 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human 11_13.E11K.R64D.S67D.R107K mature protein sequence, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8a, a human CD8 transmembrane domain, a costimulatory signal domain of cytoplasmic 4-1BB, a cleaved cytoplasmic domain of a cytokine IL2R8 (amino acid positions of 266 to 369; SEQ ID NO: 17), a mutant CD3 (positions 91 to 113 of SEQ ID NO: 18 are mutated to a sequence of SEQ ID NO: 32), T2A, a human 1L2 signal sequence, an (amino acid positions of 23 to 155; SEQ ID NO: 21) sequence, and an Xhol/Notl Date Recue/Date Received 2021-03-05 12 cleavage site (see CAR-containing polypeptide #11 of FIG. 6; and FIG. 7).
The completed structure of polypeptide CAR-containing #12 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human 11_13.E11K.R64D.S67D.R107K mature protein sequence, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a human hinge region of CD8a, a human CD8 transmembrane domain, a costimulatory signal domain of cytoplasmic 4-1BB, a cleaved cytoplasmic domain of a cytokine IL2R8 (amino acid positions of 266 to 369; SEQ ID NO: 17), a mutant CD3 (positions 91 to 113 of SEQ ID NO: 18 are mutated to a sequence of SEQ ID NO: 32), and an Xhol/Notl cleavage site (see #12 of FIG. 6; and FIG. 7).
The entire sequences of CAR-containing polypeptides #1, #2, #5, #6, #7, #8, #11, and #12 are shown in SEQ ID NOS: 23 to 30.
The finally prepared CAR gene fragments were conjugated to the MFG retrovirus expression vector digested with Xhol/Notl (Emtage PC et al., Clin Cancer Res, 2008, 14:8112-8122). In this Example, in order to compare the activity of chimeric antigen receptors, YYB103 (SEQ ID NO: 22) was further prepared.
Example 2: Preparation of CAR-T cells transformed with novel chimeric antigen receptors High-titer CAR-expressing PG13 clones were prepared such that Phoenix-Ampho and Phoenix-Eco cells were transiently transfected with the retroviral expression vector prepared in Example 1, and then, cell-free vector stocks were prepared from the transfected Phoenix-Ampho and Phoenix-Eco cells by transfecting PG13 cells.
For high-titer monoclones, PG13/#1, PG13/#2, PG13/#5, PG13/#6, PG13/#7, PG13/#8, PG13/#11, or PG13/#12 cells were stained using an anti-IL-13 monoclonal antibody (BD Pharmingen), and these single clones were isolated using a flow cytometer. The PG13/#1, PG13/#2, PG13/#5, PG13/#6, PG13/#7, PG13/#8, PG13/#11, or PG13/#12 clones were prepared by the second subcloning according to the limiting dilution method. These subclones stably showed high CAR expression and were selected for the efficient transduction ability in the peripheral blood.
The transduction degree of PG13/#1, PG13/#2, PG13/#5, PG13/#6, PG13/#7, PG13/#8, PG13/#11, or PG13/#12 cells, which were transduced using an anti-IL-monoclonal antibody (BD Pharmingen), was analyzed using a flow cytometer. The supernatants of the transduced PG13/#1, PG13/#2, PG13/#5, PG13/#6, PG13/#7, PG13/#8, PG13/#11, or PG13/#12 cells contained retrovirus, and the supernatants were collected for genetic modification of T cells.
The peripheral blood mononuclear cells (PBMCs) were separated using centrifugation by adding the whole blood obtained from a healthy human donor into Ficoll Paque (GE Healthcare). The separated PBMCs were cultured by adding an anti-CD3 monoclonal antibody (eBioscience) at a concentration of 100 ng/mL under the condition of human IL-2 (Novartis) at a concentration of 100 IU/mL to activate the T cell fraction (BL Levine, Cancer Gene Therapy, 2015, 22:79-84). Two to three days after the cultivation, most of the cells were T cells and included natural killer cells at a Date Recue/Date Received 2021-03-05 13 percentage of 0% to 2%. Two to three days after the activation step, the T
cells were subjected to transduction two times over two days using the retroviral supernatant and washed, and then proliferated for four to seven days in a flask. The cells were cultured in a stirring platform device (a WAVE bio-reactor system) for 12 to 28 days.
IL-2 was maintained at a concentration of 100 IU/mL. The T cells modified as such were used for an analysis experiment.
Experimental Example 1: Checking of growth rate and viability of CAR-T cells transformed with novel chimeric antigen receptors Experimental Results For the T cells prepared in Example 2, the number of cells was counted to confirm the growth rate and viability of CAR-T, and the results are shown in FIG. 8.
The number of cells and growth rate of all of the groups (#1, #2, #5, #6, #7, #8, #11, or #12) were shown to be higher than those of the control group (i.e., YYB103) depending on the day of the week, the cells were shown to grow rapidly from Day 12 of culture, and the viability was also shown to be 90% or higher (see FIG. 8).
Experimental Example 2: Checking of CAR expression rate on surface of CAR-T
cells transformed with novel chimeric antigen receptors Experimental Method (flow cytometric analysis) For flow cytometry (>30,000 events), a BD LSRII device (Becton Dickinson) and BD FACSDiva software (Becton Dickinson) were used. Specifically, the cells were washed once with PBS containing 2% bovine serum albumin before adding a PE-conjugated anti-human IL-13 monoclonal antibody (BD Pharmingen) thereto. After washing, the cells were reacted with each antibody at 4 C for 30 minutes in a state where light was blocked, and washed once, and thereafter, the expression rate of CAR
on the surface of transduced T cells was checked.
Experimental Results In order to confirm whether the 8 kinds of IL13Ra2-specific, CAR-containing polypeptides prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12) were expressed on the T cell surface, T cell culture was performed for 28 days according to Example 2, and then flow cytometric analysis was performed according to the experimental method.
As a result of the analysis, as shown in FIG. 9, the expression rate of the chimeric antigen receptors expressed on the cell surface of live T cells was shown to be in the range of 24.5% to 84.2%, and the expression of IL13Ra2-specific chimeric antigen receptors was stably maintained for 4 weeks, without additional activation or transduction of T cells (see FIG. 9).
Experimental Example 3: Checking of chimeric switch receptor expression rates of TGF-3R2 and IL-18R onthe cell surface of CAR-T cells transformed with novel chimeric antigen receptors Experimental Methods (flow cytometric analysis) For flow cytometry (>30,000 events), a BD LSRII device (Becton Dickinson) and BD FACSDiva software (Becton Dickinson) were used. Specifically, the cells were washed once with PBS containing 2% bovine serum albumin before adding a human TGF-8R2 fluorescein-conjugated antibody (FAB2411F) (BD Pharmingen) thereto.
After Date Recue/Date Received 2021-03-05 14 washing, the cells were reacted with each antibody at 4 C for 30 minutes in a state where light was blocked and washed once, and thereafter, the chimeric switch receptor expression rates of TGF-8R2 and IL-18R on the cell surface of transduced T
cells were checked.
Experimental Results In order to confirm whether the chimeric switch receptors of TGF8R2 and IL-18R
were expressed on the cell surface of CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12), T cell cultivation was performed for 14 days according to Example 2, and then flow cytometric analysis was performed according to the experimental method. As a result of the analysis, the expression rates of the chimeric switch receptors of TGF8R2 and IL-18R expressed on the cell surface of live T
cells were shown to be about 85% (see FIG. 10).
Experimental Example 4: Checking of phenotypes on the cell surface of CAR-T
cells transformed with novel chimeric antigen receptors Experimental Methods (flow cytometric analysis) For flow cytometry (>30,000 events), a BD LSRII device (Becton Dickinson) and a BD FACSDiva software (Becton Dickinson) were used. Specifically, the cells were washed once with PBS containing 2% bovine serum albumin before adding an FITC-conjugated CD45RA Ab (HI100) (Biolegend), a PE-conjugated CCR7Ab (G043H7) (Biolegend), and a PE-Cy7-conjugated CD62L Ab (DREG-56) (Biolegend) thereto.
After washing, the cells were reacted with each antibody at 4 C for 30 minutes in a state where light was blocked and washed once, and thereafter, the content of less-differentiated memory CAR-T cells and CCR7+CD45RA+CD62L+ phenotypes on the surface of transduced T cells were checked.
Experimental Results In order to confirm the content of the less-differentiated memory CAR-T cells on the cell surface of CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12), T cell cultivation was performed for 10 days according to Example 2, and then flow cytometric analysis was performed according to the experimental method.
As a result of the analysis, the content of less-differentiated memory CAR-T cells showing CCR7+CD45RA+CD62L+ phenotypes, which were expressed on live T cells, compared to a non-transduced sample (i.e., a control group) by 5% or higher, and compared to YYB103 (i.e., the transduced sample, CAR-T control group) by 10% or higher, on the surface of all of the groups of CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12), was confirmed (see FIG. 11).
Experimental Example 5: Checking of cytotoxicity of CAR-T cells transformed with novel chimeric antigen receptors against glioma cell line in which IL13Ra2 is overexpressed Experimental Methods In order to measure the cytotoxicity of IL13Ra2-specific CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12), cytotoxicity assay was performed using an LDH (Promega) kit. Specifically, CAR-T cells (effector cells) were used 10 days after activation of the cells with an anti-CD3 Ab, and the CAR-T cells (having a CAR
expression rate of 20% to 40%) were added to a 6-well plate at a ratio 1:2, in which Date Recue/Date Received 2021-03-05 15 effector: target was 1x106 cells : 2x106 cells and reacted at 37 C for 24 hours. As the used target cells, U87 cells (expressing IL13Ra2) and 293FT cells (the control group for normal cells) were used.
Experimental Results An analysis was performed to examine whether IL13Ra2-specific CAR-T cells prepared in the present invention can effectively kill the target cancer cells (U87). The method used was such that where target cancer cells (U87) and normal cells (293FT) described above were cultured together with each of the activated CAR-T cells and cytotoxicity was compared and analyzed. As shown in FIG. 12, all of the CAR-T
cells prepared according to the present invention showed a higher effect by 50% to 70% of inducing the death of the target cancer cell (U87) compared to non-transduced activated T cells. In particular, CAR-T #5, #6, and #11 of the present invention showed high cytotoxicity. In addition, CAR-T cells (#1, #2, #5, and #6) containing IL-7Ra exhibited higher overall cytotoxicity compared to CAR-T cells (#7, #8, #11, and #12) containing IL-21R8.
From the experimental result where 293FT cells, which do not express IL13Ra2, were used as the control group for normal cells, as an object to be compared with the target cells, it was confirmed that the CAR-T cells specific to IL13Ra2 showed very weak cytotoxicity (2% to 4%). This result shows that the chimeric antigen receptors used in this experiment bind specifically to IL13Ra2.
Through this Experimental Example, it was found that IL13Ra2-specific chimeric antigen receptor T cells do not show cytotoxicity to a normal cell (293FT) and significantly kill target cancer cells (U87), which express IL13Ra2.
FIG. 13 shows the results in which the cytotoxicity of the CAR-T cells, which were transformed with the chimeric antigen receptor (CAR) of the present invention, was confirmed through LDH assay after 24 hours or 96 hours without co-culture with target cells. Since the spontaneous toxicity of YYB103 is higher than that of other groups, the 8 groups are expected to show higher cytotoxicity compared to YYB103 in the co-culture with U87 cells, and the CAR-T cells (#1, #2, #5, #6, #7, #8, #11, or #12), which were transformed with the chimeric antigen receptor (CAR) of the present invention, are expected to have a comparative advantage in terms of safety and stability.
Experimental Example 6: Checking of changes in CAR expression rate when CAR-T cells, which were transformed with novel chimeric antigen receptors, are co-cultured with gliorna cell line in which IL13Ra2 is overexpressed Experimental Methods In order to check the changes in CAR expression rate when the CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12) are co-cultured with glioma cell line in which IL13Ra2 is overexpressed, the CAR-T cells (effector cells) having a CAR expression rate of 20% to 40% were used 10 days after activation of the cells with an anti-CD3 Ab. The CAR-T cells were added to a 6-well plate at a ratio 1:2, in which effector: target was 1x106 cells: 2x106 cells and reacted at 37 C for 24, 48, and 96 hours. To the sample, which was reacted for 96 hours, were added 2x106 cells of the target cells after 48 hours, and a BD LSRII device (Becton Dickinson) and BD
FACSDiva software (Becton Dickinson) were used for flow cytometry (>30,000 events).
Date Recue/Date Received 2021-03-05 16 Specifically, the cells were washed once with PBS containing 2% bovine serum albumin before adding a PE-conjugated anti-human IL-13 monoclonal antibody (BD
Pharmingen) thereto. After washing, the cells were reacted with each antibody at 4 C for 30 minutes in a state where light was blocked and washed once, and thereafter, the changes in the CAR expression rate were checked.
Experimental Results In order to confirm the changes in the CAR expression rate when the 8 kinds of IL13Ra2-specific CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12) are co-cultured with glioma cell line in which IL13Ra2 is overexpressed, the T cells were cultured for 10 days, and then flow cytometric analysis was performed according to the experimental method according to Example 2. As a result of the analysis, as shown in FIG. 14, the CAR-T cells which were co-cultured with U87 cells for 24 hours showed a smaller change in CAR expression in all of the 8 groups compared to that of YYB103 (FIG. 14).
In co-culture with U87 cells for 48 hours, the change in CAR expression was shown to be smaller than that of YYB103 in all of the 8 groups (FIG. 15).
In co-culture with U87 cells for 96 hours, the change in CAR expression was shown to be smaller than that of YYB103 in 5 groups (i.e., #1, #2, #5, #6, and #7) (FIG. 16). The CAR expression of YYB103 after co-culture with U87 cells for 96 hours was shown to be insignificant (FIG. 16).
The changes in CAR expression during the co-culture with glioma cell line, in which IL13Ra2 is overexpressed, the changes in the CAR expression rate without co-culture with target cells or during the co-culture with 293FT cells (which were used as the control group for normal cells) can confirm that the chimeric antigen receptor (CAR) according to the present invention is expected to show excellent in vivo persistence and anti-tumor effects of CAR-T cells in a hostile tumor microenvironment, while simultaneously reducing toxicity.
Experimental Example 7: Checkind of chandes in chimeric switch receptor expression rate of TGFOR2 and IL-18R when CAR-T cells which were transformed with novel chimeric antiden receptors are co-cultured with dlioma cell line, in which IL13Ra2 is overexpressed Experimental Methods In order to check the changes in the chimeric switch receptor expression rate of TGF3R2 and IL-18R when CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12) are co-cultured with glioma cell line, in which IL13Ra2 is overexpressed, the CAR-T cells (effector cells) having a CAR expression rate of 20% to 40%
were used days after activation of the cells with an anti-CD3 Ab. The CAR-T cells were added to a 6-well plate at a ratio 1:2, in which effector: target was 1x106 cells:
2x106 cells and reacted at 37 C for 24, 48, and 96 hours. To the sample, which was reacted for hours, were added 2x106 cells of the target cells after 48 hours, and a BD
LSRII device (Becton Dickinson) and BD FACSDiva software (Becton Dickinson) were used for flow cytometry (>30,000 events). Specifically, the cells were washed once with PBS
containing 2% bovine serum albumin before adding an anti-human TGF-31R2 fluorescein-conjugated antibody (FAB2411F) (BD Pharmingen) thereto. After washing, Date Recue/Date Received 2021-03-05 17 the cells were reacted with each antibody at 4 C for 30 minutes in a state where light was blocked and washed once, and thereafter, the changes in the chimeric switch receptor expression rate of TGF8R2 and IL-18R were checked.
Experimental Results In order to confirm the changes in the chimeric switch receptor expression rate of TGF8R2 and IL-18R when the 8 kinds of IL13Ra2-specific CAR prepared in Example
2 (#1, #2, #5, #6, #7, #8, #11, or #12) are co-cultured with glioma cell line in which IL13Ra2 is overexpressed, the T cells were cultured for 10 days according to Example 2, and then flow cytometric analysis was performed according to the experimental method.
As a result of the analysis, as shown in FIG. 17, the expression of the chimeric switch receptors of TGF8R2 and IL-18R in the 4 groups (#1, #2, #7, and #8) after 24 hours of co-culture showed a tendency of a decrease in the groups co-cultured with glioma cell line, U87 cells compared to those cultured with CAR-T cells only, but the average expression rate was 30% (FIG. 17). The expression of the chimeric switch receptors of TGF8R2 and IL-18R in the 4 groups (#1, #2, #7, and #8) after 48 hours of co-culture showed a tendency of a decrease in the groups co-cultured with U87 cells compared to those cultured with CAR-T cells only, but the average expression rate was 25%. The expression of the chimeric switch receptors of TGF8R2 and IL-18R in the 4 groups (#1, #2, #7, and #8) after 72 hours of co-culture showed a tendency of a decrease in the groups co-cultured with U87 cells compared to those cultured with CAR-T cells only, but the average expression rate was 15%. Although the changes in the expression rate were greater compared to one without co-culture with target cells or the co-culture with 293FT cells (which were used as the control group for normal cells), by the confirmation of the results of the chimeric switch receptor expression rate of TGF8R2 and IL-18R against glioma cell line in which IL13Ra2 is overexpressed, the chimeric antigen receptor (CAR) according to the present invention is expected to show excellent in vivo persistence and anti-tumor effects of CAR-T cells in a hostile tumor microenvironment.
Experimental Example 8: Checking of cytokine (IFN-v) production by T cells, which were transformed with novel chimeric antigen receptors, against target cells Experimental Methods In order to confirm the cytokine (IFN-y) production after 24 hours and 48 hours of co-culture between the CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12) and glioma cell line (U87 cells) and 293FT target cells (which were used as a control group for normal cells), the CAR-T cells (effector cells) in which the CAR
expression rate is in the range of 20% to 40% were used 10 days after activation of the cells with an anti-CD3 Ab. The CAR-T cells were added to a 6-well plate at a ratio in which effector: target was 1x106 cells: 2x106 cells, 6 mL of a culture medium was added per well, and the cells reacted at 37 C for 24 hours. 100 pL of the supernatant was collected and transferred to a 1.5 mL tube, the cells were cultured further up to 48 hours, and 100 pL of the supernatant was collected from the culture in the same manner.
The IFN-y analysis experiment was performed as follows according to the manufacturer's instructions of the ELISA analyzer (R&D systems). 3 mL of the Date Recue/Date Received 2021-03-05 18 calibrator diluent RD6-21 was added to an IFN-y standard bottle and mixed in a shaker for 15 minutes, dispensed in an amount of 1 mL per 1.5 mL tube to prepare two "standard rs. 500 pL was taken from 1 mL of the "standard 1" dispensed and was subjected to serial dilution to prepare up to "standard 7". In order to prepare blanks, 500 pL of the culture medium and 500 pL of the calibrator diluent RD6-21 were mixed.
In order to dilute the samples by 1/20, 190 pL of the calibrator diluent RD6-was added into each of new 1.5 mL tubes as many as the number of samples. The assay samples were each prepared to a total volume of 200 pL by collecting 10 pL of the supernatant of each sample. To prepare a wash buffer, 500 mL of distilled water and 20 mL of wash buffer concentrate were mixed well in a 500 mL storage bottle.
Assay diluent RD1-51 (blue dye) in an amount of 100 pL each was added to the IFN-y microplate for each well of "standard 1" to "standard 7", blank, and samples. After adding 100 pL each of the blank, standards, and samples prepared above, a plate sealer was attached thereto and reacted at room temperature for 2 hours. After the reaction, the reaction solution was discarded, and 400 pL of the prepared wash buffer was added thereto, and the wells were washed 4 times. 200 pL of IFN-y conjugate was added to each well, a plate sealer was attached thereto, and the reaction was performed at room temperature for 2 hours. The reaction solution was discarded, and the wells were washed 4 times using 400 pL of the wash buffer. After mixing the color reagent A: B in a 1:1 ratio, the mixture was added in an amount of 200 pL per well, a plate sealer was attached thereto, the light was blocked with foil, and the reaction was performed at room temperature for 30 minutes. After the reaction, 50 pL of the stop solution was added to each well and measured at 450 nm within 30 minutes.
Experimental Results YYB103 was shown to secrete a relatively high amount of IFN-y compared to other samples. When CAR-T cells were cultured in the presence of U87 cells, CAR-T
(#1, #2, #5, and #6) containing an IL-7Ra signaling domain were shown to contain more IFN-y compared to CAR-T (#7, #8, #11, and #12) containing an IL-21R8 signaling domain. Results between samples after 24 hours and 48 hours of incubation showed similar patterns (FIG. 18).
As shown in FIG. 12, when the cytotoxicity was confirmed through LDH assay after 24 hours of co-culture between the CAR-T (which were transformed with the chimeric antigen receptor (CAR) of the present invention), U87 cells (human glioma cell line), and 293FT target cells (which are used as a control group for normal cells), the 8 groups showed similar abilities of killing target cells. However, considering that the production of IFN-y was small and thus target cells could be killed without adverse effects caused by cytokines (cytokine release syndrome, CRS), in terms of safety, the effect of the CAR-T cell therapeutic is expected to have a fewer side effects while having excellent therapeutic effects.
Experimental Example 9: Checking of cytokine (IL-21) production by CAR-T
cells, which were transformed with novel chimeric antigen receptors Experimental Methods In order to confirm the cytokine (IL-21) production after 24 hours and 48 hours of co-culture between the CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, Date Recue/Date Received 2021-03-05 19 or #12) and glioma U87 cells and 293FT target cells (which were used as a control group for normal cells), the CAR-T cells (effector cells) in which the CAR
expression rate is in the range of 20% to 40% were used 10 days after activation of the cells with an anti-CD3 Ab. The CAR-T cells were added to a 6-well plate at a ratio in which effector: target was 1x106 cells: 2x106 cells, 6 mL of a culture medium was added per well, and the cells were reacted at 37 C for 24 hours. Then, 100 pL of the supernatant was collected and transferred to a 1.5 mL tube, the cells were cultured further up to 48 hours, and 100 pL of the supernatant was collected from the culture in the same manner.
The IL-21 analysis experiment was performed as follows according to the manufacturer's instructions of the ELISA analyzer (Invitrogen). 100 pL of a capture antibody was added to each well of a 96-well ELISA plate and reacted at 4 C
overnight.
After the reaction, the wells were washed 3 times using 250 pL of wash buffer.
ELISA/ELISASPOT diluent was added in an amount of 200 pL per well and reacted at room temperature for one hour.
After the reaction, 100 pL each of the prepared blank, cytokine IL-21 standard, and samples were added thereto, and ELISA/ELISASPOT diluent was added in an amount of 100 pL per well, and a plate sealer was attached thereto and the reaction was performed at room temperature for two hours. After the reaction, the wells were washed 3 times using 250 pL of wash buffer. 100 pL of a detection antibody was added to each well, and a plate sealer was attached thereto and the reaction was performed at room temperature for one hour. After the reaction, the wells were washed 3 times using 250 pL of wash buffer. 100 pL of Avidin-HRP was added to each well, a plate sealer was attached thereto, and the reaction was performed at room temperature for minutes. After the reaction, the wells were washed 5 times using 250 pL of wash buffer.
100 pL of lx TMB solution was added to each well, a plate sealer was attached thereto, and the reaction was performed at room temperature for 30 minutes. After the reaction, 50 pL of the stop solution was added to each well and measured at 450 nm within 30 minutes.
Experimental Results It was confirmed that IL-21 was secreted in IL-21 gene-containing CAR-T cells (#1, #5, #7, and #11). When the CAR-T cells were co-cultured with U87 cells, a greater amount of IL-21 was secreted, and considering that the overall experimental results at 24 hours and 48 hours were similar, no significant difference according to the incubation time was observed. As the result of the experiment at 48 hours showed a higher concentration than that at 24 hours, the secretion of IL-21 appeared to proceed steadily (FIG. 19).
The secretion of IL-21 in an environment of cancer cells is speculated to increase the ability to kill cancer cells by assisting the activation of innate immune-related cells.
Example 3: Preparation of cancer antigen EGFRvIll targeting CAR vector and aV3 targeting CAR vector In the case of EGFRvIll, which is a major antigen for tumors such as glioblastoma and lung cancer, in order to reduce the side effects of CAR-T
cells through non-specific binding while minimizing the binding affinity for EGFR wild-type in which Date Recue/Date Received 2021-03-05 20 the specificity for EGFRvIll is maintained, in SEQ ID NO: 33, positions 52 to 57 of SEQ
ID NO: 3 were first changed from STGGYN to DPENDE (a CDR2 part of a heavy chain), position 101 of SEQ ID NO: 3 was changed from S to G (a CDR3 part of a heavy chain), and position 229 of SEQ ID NO: 3 was changed from V to G (a CDR3 part of a light chain).
Human CD3 (P20963-1), human CD8A (P01732), human 4-1BB (Q07011), human CD3Z (P20963), a Gaussia princeps luciferase, and human kappa light chain signal sequence (HuVHCAMP) were optimized using scientific literature and publicly available databases, and thereby chimeric antigen receptor-containing polypeptides consisting of codon-optimized synthetic DNA (#13, #14, #15, and #16 of SEQ ID
NOS: 34 to 37) were prepared.
The completed structure of CAR-containing polypeptide #13 includes a Kozak consensus ribosome-binding sequence, a CD8A signal sequence, an antigen binding domain which binds to EGFRvIll of SEQ ID NO: 3, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8A, a human 0D8 transmembrane domain, a cytoplasmic 4-1BB costimulatory signal domain, a CD3 cytoplasmic domain (SEQ ID NO: 18), and an Xhol/Notl cleavage site.
The finally prepared CAR gene fragment was conjugated to the MFG retrovirus expression vector digested with Xhol/Notl (Emtage PC et al., Clin Cancer Res, 2008, 14:8112-8122). In this Example, in order to compare the activity of chimeric antigen receptors, YYB105 (SEQ ID NO: 39; see PCT International Publication No. WO 2017/023138) was additionally prepared.
The completed structure of CAR-containing polypeptide #14 includes a Kozak consensus ribosome-binding sequence, a CD8A signal sequence, an antigen binding domain which binds to EGFRvIll of SEQ ID NO: 32, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8A, a human 0D8 transmembrane domain, a cytoplasmic 4-1BB costimulatory signal domain, a CD3 cytoplasmic domain (SEQ ID NO: 18), and an Xhol/Notl cleavage site. The finally prepared CAR gene fragment was conjugated to the MFG retrovirus expression vector digested with Xhol/Notl (Emtage PC et al., Clin Cancer Res, 2008,14:8112-8122).
In the case of aV8 anti-angiogenic, the CAR expression rate and persistence in CAR-T cells targeting these were optimized. In the case of aVp, the CAR
expression rate and persistence in CAR-T cells targeting these were optimized using Gaussia princeps luciferase signal sequence or 0D8 signal sequence (SEQ ID NO: 36 and SEQ
ID NO: 37).
The completed structure of CAR-containing polypeptide #15 includes a Kozak consensus ribosome-binding sequence, a Gaussia princeps luciferase signal sequence, SEQ ID NO: 5 (an antigen binding domain which binds to aV8), three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a Date Recue/Date Received 2021-03-05 21 CAR protein, a hinge region of human CD8A, a human 0D8 transmembrane domain, a cytoplasmic 4-1BB costimulatory signal domain, a CD3 cytoplasmic domain (SEQ
ID
NO: 18), and an Xhol/Notl cleavage site. The finally prepared CAR gene fragment was conjugated to the MFG retrovirus expression vector digested with Xhol/Notl (Emtage PC etal., Clin Cancer Res, 2008, 14:8112-8122). In this Example, in order to compare the activity of chimeric antigen receptors, YYB107 (SEQ ID NO: 38; see PCT
International Publication No. WO 2017/023138) was additionally prepared.
The completed structure of CAR-containing polypeptide #16 includes a Kozak consensus ribosome-binding sequence, a Gaussia princeps luciferase signal sequence, SEQ ID NO: 5 (an antigen binding domain which binds to aV8), three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8A, a human 0D8 transmembrane domain, a cytoplasmic 4-1BB costimulatory signal domain, a CD3 cytoplasmic domain (SEQ
ID
NO: 18), and an Xhol/Notl cleavage site. The finally prepared CAR gene fragment was conjugated to the MFG retrovirus expression vector digested with Xhol/Notl (Emtage PC etal., Clin Cancer Res, 2008, 14:8112-8122).
Example 4: Preparation of CAR-T cells transformed with chimeric antigen receptors High-titer CAR-expressing PG13 clones were prepared such that Phoenix-Ampho and Phoenix-Eco cells were transiently transfected with the retroviral expression vector prepared in Example 1, and then, cell-free vector stocks were prepared from the transfected Phoenix-Ampho and Phoenix-Eco cells by transfecting PG13 cells.
For high-titer monoclones, PG13/#13, PG13/#14, PG13/#15, and PG13/#16 cells were stained using an anti-myc Ab (BD Pharmingen), and these monoclones were isolated using a flow cytometer. The transduction degree of PG13/#13, PG13/#14, PG13/#15, and PG13/#16 cells, which were transduced using an anti-myc Ab, was analyzed using a flow cytometer. The supernatants of the transduced PG13/#13, PG13/#14, PG13/#15, and PG13/#16 cells contained retrovirus, and the supernatants were collected for genetic modification of T cells. The peripheral blood mononuclear cells (PBMCs) were separated using centrifugation by adding the whole blood obtained from a healthy human donor into Ficoll Paque (GE Healthcare). The separated PBMCs were cultured by adding an anti-CD3 monoclonal antibody (eBioscience) at a concentration of 100 ng/mL under the condition of human IL-2 (Novartis) at a concentration of 100 IU/mL
to activate the T cell fraction (BL Levine, Cancer Gene Therapy, 2015, 22:79-84). Two to three days after the cultivation, most of the cells were T cells and included natural killer cells at a percentage of 0% to 2%. Two to three days after the activation step, the T cells were subjected to transduction two times over two days using the retroviral supernatant and washed, and then proliferated for 14 days in a flask. IL-2 was maintained at a concentration of 100 IU/mL. The T cells modified as such were used for an analysis experiment.
Experimental Example 1: Checking of growth rate and viability of CAR-T cells transformed with chimeric antigen receptors Experimental Results Date Recue/Date Received 2021-03-05 22 For the T cells prepared in Example 4 above, the number of cells was counted to confirm the growth rate and viability rate of CAR-T, and the results are shown in FIG. 20.
The number of cells and growth rate of all of the groups (#13, #14, #15, and #16) were shown to be similar to those of the control group (i.e., YYB105) depending on the day of the week, the cells were shown to grow rapidly from Day 12 of culture, and the viability was also shown to be 90% or higher (see FIG. 20).
Experimental Example 2: Checking of CAR expression rate on the cell surface of CAR-T cells transformed with chimeric antigen receptors Experimental Methods (flow cytometric analysis) For flow cytometry (>30,000 events), a BD LSRII device (Becton Dickinson) and BD FACSDiva software (Becton Dickinson) were used. Specifically, the cells were washed once with PBS containing 2% bovine serum albumin before adding a PE-conjugated anti-myc antibody (BD Pharmingen) thereto. After washing, the cells were reacted with each antibody at 4 C for 30 minutes in a state where light was blocked and washed once, and thereafter, the expression rate of CAR on the surface of transduced T cells was checked.
Experimental Results In order to confirm whether the 4 kinds of CAR prepared in Example 2 (#13, #14, #15, and #16) were expressed on the T cell surface, T cell cultivation was performed for 14 days according to Example 4, and then flow cytometric analysis was performed according to the experimental method.
As a result of the analysis, as shown in FIG. 21, the expression rate of the chimeric antigen receptors expressed on the surface of live T cells was shown to increase in #13 (43.0%) and #14 (50.8%) compared to YYB105 (37.4%) (i.e., a control group) (FIG. 21); and was shown to increase in #15 (45.9%) and #16 (40.0%) compared to YYB107 (24.6%) (i.e., a control group) (FIG. 21).
[Industrial Applicability]
The present invention relates to rapidly developing a CAR-T cell in the field of cancer treatment. The CAR-T cell according to the present invention has a remarkably excellent expression rate and persistence, and thus has an improved therapeutic effect for solid cancer, etc., and can be effectively used in the field of customized cancer treatment.
[SEQUENCE LISTING]
SEQ ID NO: 1 {antigen binding wild type IL13 domain binding to IL13Ra2}
Length: 112 Type: ligand protein Organism: human Sequence:
GPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAG
MYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSL
HVRDTKIEVAQFVKDLLLHLKKLFREGQFN
Date Recue/Date Received 2021-03-05 23 SEQ ID NO: 2 {antigen binding domain capable of binding to an antigen associated with an angiogenic activity}
Length: 92 Type: ligand protein Organism: human Sequence:
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFT
VLQPPSTATISGLKPGVDYTITVYAVVERNGRELNTPPISINY
RTHHHHHH
SEQ ID NO: 3 {antigen binding domain binding to EGFRvIll}
Length: 252 Type: scFv protein Organism: human Sequence:
QVQLQESGGGLVKPGGSLKLSCAASGFTFSKFGMSWVRQT
PDKRLEWVASISTGGYNTFYSDNVKGRFTISRDNAKNTLYL
QMSSLKSEDTAMYYCARGYSSTSFAMDYWGQGTMVTVSSG
STSGSGKPGSGEGSDIQMTQSPSSLSASVGDRVTITCMTST
DIDDDMNWYQQKPGKTPKLLIYEGNTLRPGVPSRFSGSGSG
TDFIFTISSLQPEDIATYYCLQSFNVPLTFGGGTKVEIKEQKL
ISEEDL
SEQ ID NO: 4 {antigen binding domain binding to EphA2}
Length: 141 Type: ligand protein Organism: human Sequence:
DRHTVFWNSSNPKFRNEDYTIHVQLNDYVDIICPHYEDHSV
ADAAMEQYILYLVEHEEYQLCQPQSKDQVRWQCNRPSAKH
GPEKLSEKFQRFTAFALAKEFKAGHSYYYISKPIHQHEDRCL
RLKVTVSGEQKLISEEDL
SEQ ID NO: 5 {antigen binding domain binding to aVp}
Length: 104 Type: ligand protein Organism: human Sequence:
VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGG
NSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTPRGDWN
EGSKPISINYRTEQKLISEEDL
SEQ ID NO: 6 {antigen binding domain binding to glypican1}
Date Recue/Date Received 2021-03-05 24 Length: 418 Type: ligand protein Organism: human Sequence:
TSPCDNFDCQNGAQCIVRINEPICQCLPGYQGEKCEKLVSV
NFINKESYLQIPSAKVRPQTNITLQIATDEDSGILLYKGDKDH
lAVELYRGRVRASYDTGSHPASAIYSVETINDGNFHIVELLA
LDQSLSLSVDGGNPKIITNLSKQSTLNFDSPLYVGGMPGKS
NVASLRQAPGQNGTSFHGCIRNLYINSELQDFQKVPMQTGI
LPGCEPCHKKVCAHGTCQPSSQAGFTCECQEGWMGPLCD
QRTNDPCLGNKCVHGTCLPINAFSYSCKCLEGHGGVLCDEE
EDLFNPCQAIKCKHGKCRLSGLGQPYCECSSGYTGDSCDR
EISCRGERIRDYYQKQQGYAACQTTKKVSRLECRGGCAGG
QCCGPLRSKRRKYSFECTDGSSFVDEVEKVVKCGCTRCVS
EQKLISEEDL
SEQ ID NO: 7 {antigen binding domain binding to mesothelin}
Length: 262 Type: scFv protein Organism: human Sequence:
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSG
STNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGKNGAFDIWGQGTMV
TVSSGSTSGSGKPGSGEGSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWN
WI RQSPS RGLEWLGRTYYRSKVVYN DYAVSVKSRMS IN PDTSKNQFSLQLNSVTP
EDTAVYYCARGMMTYYYGMDV WGQGTTVTVSSGILGS
SEQ ID NO: 8 {hinge region sequence -1}
Length: 47 Type: protein Organism: human Sequence:
KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG
LDFACD
SEQ ID NO: 9 {hinge region sequence -2}
Length: 45 Type: protein Organism: human Sequence:
KPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVHTRG
LDFA
SEQ ID NO: 10 {transmembrane domain sequence-1}
Date Recue/Date Received 2021-03-05 25 Length: 21 Type: protein Organism: human Sequence:
IYIWAPLAGTCGVLLLSLVIT
SEQ ID NO: 11 {transmembrane domain sequence-2}
Length: 23 Type: protein Organism: human Sequence:
LAYLLDGILFIYGVILTALFLRV
SEQ ID NO: 12 {4-1BB}
Length: 42 Type: protein Organism: human Sequence:
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE
L
SEQ ID NO: 13 {Wild type CD28}
Length: 41 Type: protein Organism: human Sequence:
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYR
S
SEQ ID NO: 14 {IL-7Ra}
Length: 459 Type: protein Organism: human Sequence:
MTILGTTFGMVFSLLQVVSGESGYAQNGDLEDAELDDYSFSCYSQLEVNGSQHSLTC
AFEDPDVNITNLEFEICGALVEVKCLNFRKLQEIYFIETKKFLLIGKSNICVKVGEKSLTCK
KIDLTTIVKPEAPFDLSVVYREGANDFVVTFNTSHLQKKYVKVLMHDVAYRQEKDENK
VVTHVNLSSTKLTLLQRKLQPAAMYEIKVRSIPDHYFKGFWSEWSPSYYFRTPEINNSS
GEMDPILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVS
FNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV
VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNS
TLPPPFSLQSGILTLNPV AQGQPILTSLGSNQEEAYVTMSSFYQNQ
SEQ ID NO: 15 {part of IL-7Ra}
Date Recue/Date Received 2021-03-05 26 Length: 64 Type: protein Organism: human Sequence:
KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGF
LQDTF
SEQ ID NO: 16 {IL-2R8}
Length: 551 Type: protein Organism: human Sequence:
MAAPALSWR LP LLI LLLPLATSWASAAVNGTSQFTCFYNS RAN ISCVWSQDGALQDTS
CQVHAWPDRRRWNQTCELLPVSQASWACN LI LGAPDSQKLTTVDIVTLRVLCREGVR
WRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSP
GHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRT
KPAALGKDTI PWLGH LLVGLSGAFGFI I LVYLLI NCRNTGPWLKKVLKCNTPDPSKFFSQ
LSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASL
SSNHSLTSCFTNQGYFFFHLPDALEI EACQVYFTYDPYSE EDPDEGVAGAPTGSS PQP
LQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVP
RDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQG
EFRALNARLPLNTD AYLSLQELQGQDPTHLV
SEQ ID NO: 17 {part of IL-2R8}
Length: 104 Type: protein Organism: human Sequence:
NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEI
SPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHL
SEQ ID NO: 18 {CD3}
Length: 113 Type: protein Organism: human Sequence:
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG
RDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGE
RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SEQ ID NO: 19 {TGF-8R2 exodomain}
Length: 137 Type: protein Organism: human Date Recue/Date Received 2021-03-05 27 Sequence:
TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKP
QEVCVAVWRKNDENITLETVCHDPKLPYHDFI LEDAASPKCI MKEKKKPGETFFMCSC
SSDECNDNIIFSEEYNTSNPD
SEQ ID NO: 20 {transmembrane domain and endodomain of IL-18R}
Length: 219 Type: protein Organism: human Sequence:
ECRPENGEEHTFAVEILPRVLEKHFGYKLCIFERDVVPGGAVVDEIHSLIEKSRRLIIVLS
KSYMSNEVRYELESGLHEALVERKI KI I LI EFTPVTDFTFL
PQSLKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPV LSES
SEQ ID NO: 21 {IL-21}
Length: 133 Type: protein Organism: human Sequence:
QGQDRH MI RM RQLI DIVDQLKNYVN DLVPE FLPAPE DVETNCEWSAFSCFQKAQLKSA
NTGNNERI I NVSI KKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK
EFLERFKSLLQKMIHQHLSSRTHGSEDS
SEQ ID NO: 22 {YYB 103}
Length: 359 Type: protein Organism: human Sequence:
MGWSCIILFLVATATGVHSGPVPPSTALRKLIEELVNITQNQ
KAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQD
MLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKL
FKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEAC
RPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRG
RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRV
KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD
PEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR
RGKGHDGLYQGLSTATKDTYDALHMQALPPR
SEQ ID NO: 23 {#1}
Length: 998 Type: protein Organism: human Sequence:
Date Recue/Date Received 2021-03-05 28 MGWSCI I LFLVATATGVHSGPVPPSTALRKLI EE LVN ITQNQKAPLCNGS MVWS IN LTA
GMYCAALESLINVSGCSAIEKTQDMLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLL
LHLKKLFKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR
GLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYI FKQPFM RPVQTTQE EDGCSCR
FPEEEEGGCELKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVD
DIQARDEVEGFLQDTFRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR
DPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYLPQSTA
TKDTYDYVTMQALPPREGRGS LLTCGDVEEN PGPMALPVTALLLPLALLLHAARPTI PP
HVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEV
CVAVWRKNDE NITLETVCHDPKLPYHDFI LE DAASPKCI MKEKKKPGETFFMCSCSSD
DETLTDGKTYDAFVSYLKECRPENGEEHTFAVEI LPRVLEKHFGYKLCI FE RDVVPGGA
VVDE I HSLI EKSRRLI IVLSKSYMSNEVRYELESGLHEALVERKIKI I LI EFTPVTDFTFLPQ
SLKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPVLSESRRKR
SGSGATNFSLLKQAGDVEE NPGPMYRMQLLSCIALSLALVTNSQGQDRH MI RMRQLI D
IVDQLKNYVN DLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGN N ERI I NVSI KKL
KRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSE
DS
SEQ ID NO: 24 {#2}
Length: 818 Type: protein Organism: human Sequence:
MGWSCI I LFLVATATGVHSGPVPPSTALRKLI EE LVN ITQNQKAPLCNGS MVWS IN LTA
GMYCAALESLINVSGCSAIEKTQDMLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLL
LHLKKLFKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR
GLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYI FKQPFM RPVQTTQE EDGCSCR
FPEEEEGGCELKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVD
DIQARDEVEGFLQDTFRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR
DPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYLPQSTA
TKDTYDYVTMQALPPREGRGS LLTCGDVEEN PGPMALPVTALLLPLALLLHAARPTI PP
HVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEV
CVAVWRKNDE NITLETVCHDPKLPYHDFI LE DAASPKCI MKEKKKPGETFFMCSCSSD
DETLTDGKTYDAFVSYLKECRPENGEEHTFAVEI LPRVLEKHFGYKLCI FE RDVVPGGA
VVDE I HSLI EKSRRLI IVLSKSYMSNEVRYELESGLHEALVERKIKI I LI EFTPVTDFTFLPQ
SLKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKT VKPGRDEPEVLPVLSES
SEQ ID NO: 25 {#5}
Length: 594 Type: protein Organism: human Sequence:
Date Recue/Date Received 2021-03-05 29 MGWSCI I LFLVATATGVHSGPVPPSTALRKLI EE LVN ITQNQKAPLCNGS MVWS IN LTA
GMYCAALESLINVSGCSAIEKTQDMLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLL
LHLKKLFKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR
GLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYI FKQPFM RPVQTTQE EDGCSCR
FPEEEEGGCELKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVD
DIQARDEVEGFLQDTFRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR
DPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYLPQSTA
TKDTYDYVTMQALPPREGRGSLLTCGDVEENPGPMYRMQLLSCIALSLALVTNSQGQ
DRH MI RM RQLI DIVDQLKNYVN DLVPEFLPAPE DVETNCEWSAFSCFQKAQLKSANTG
NNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIH
QH LSSRTHGSEDS
SEQ ID NO: 26 {#6}
Length: 423 Type: protein Organism: human Sequence:
MGWSCI I LFLVATATGVHSGPVPPSTALRKLI EE LVN ITQNQKAPLCNGS MVWS IN LTA
GMYCAALESLINVSGCSAIEKTQDMLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLL
LHLKKLFKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR
GLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYI FKQPFM RPVQTTQE EDGCSCR
FPEEEEGGCELKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVD
DIQARDEVEGFLQDTFRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR
DPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYLPQSTA
TKDTYDYVTMQALPPR
SEQ ID NO: 27 {#7}
Length: 1038 Type: protein Organism: human Sequence:
KMIHQHLSSRTHGSEDS
SEQ ID NO: 28 {#8}
Length: 858 Type: protein Organism: human Sequence:
MGWSCI I LFLVATATGVHSGPVPPSTALRKLI EE LVN ITQNQKAPLCNGS MVWS IN LTA
GMYCAALESLINVSGCSAIEKTQDMLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLL
LHLKKLFKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR
GLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYI FKQPFM RPVQTTQE EDGCSCR
FPEEEEGGCELNCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPS
SSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHL
Date Recue/Date Received 2021-03-05 30 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNP
QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYLSLSTATKDTYLPQHMQALPP
REGRGSLLTCGDVE EN PGPMALPVTALLLPLALLLHAARPTI PPHVQKSVN N DM IVTDN
NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLET
VCHDPKLPYHDFI LE DAASPKCI MKEKKKPGETFFMCSCSSDECNDNI I FSE EYNTSNP
DPGHVFTRG M I IAVLI LVAVVCLVTVCVIYRVDLVLFYR H LTR R DETLTDGKTYDAFVSY
LKECRPENGEEHTFAVEI LPRVLEKHFGYKLCI FERDVVPGGAVVDEI HSLI EKSRRLI IV
LSKSYMS N EVRYELESGLH EALVE RKI KI I LI EFTPVTDFTFLPQSLKLLKSH RVLKWKAD
KSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPVLSES
SEQ ID NO: 29 {#11}
Length: 634 Type: protein Organism: human Sequence:
MGWSCI I LFLVATATGVHSGPVPPSTALRKLI EE LVN ITQNQKAPLCNGS MVWS IN LTA
GMYCAALESLINVSGCSAIEKTQDMLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLL
LHLKKLFKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR
GLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYI FKQPFM RPVQTTQE EDGCSCR
FPEEEEGGCELNCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPS
SSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHL
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNP
QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYLSLSTATKDTYLPQHMQALPP
REGRGSLLTCGDVE ENPGPMYRMQLLSCIALSLALVTNSQGQDRH MI RMRQLI DIVDQ
LKNYVN DLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGN N ERI I NVS I KKLKRKP
PSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS
SEQ ID NO: 30 {#12}
Length: 463 Type: protein Organism: human Sequence:
MGWSCI I LFLVATATGVHSGPVPPSTALRKLI EE LVN ITQNQKAPLCNGS MVWS IN LTA
GMYCAALESLINVSGCSAIEKTQDMLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLL
LHLKKLFKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR
GLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYI FKQPFM RPVQTTQE EDGCSCR
FPEEEEGGCELNCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPS
SSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHL
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNP
QEGLYNELQKDKMAEAYSEI
GMKGERRRGKGHDGLYLSLSTATKDTYLPQHMQALPPR
SEQ ID NO: 31 {mutated 3rd ITAM-1}
Length: 23 Date Recue/Date Received 2021-03-05 31 Type: protein Organism: human Sequence:
YLPQSTATKDTYDYVTMQALPPR
SEQ ID NO: 32 {mutated 3rd ITAM-2}
Length: 23 Type: protein Organism: human Sequence:
YLSLSTATKDTYLPQHMQALPPR
SEQ ID NO: 33 {antigen binding domain binding to EGFRvIll}
Length: 252 Type: scFv protein Organism: human Sequence:
QVQLQESGGGLVKPGGSLKLSCAASGFTFSKFGMSWVRQT
PDKRLEWVASIDPENDETFYSDNVKGRFTISRDNAKNTLYL
QMSSLKSEDTAMYYCARGYGSTSFAMDYWGQGTMVTVSS
GSTSGSGKPGSGEGSDIQMTQSPSSLSASVGDRVTITCMTS
TDIDDDMNWYQQKPGKTPKLLIYEGNTLRPGVPSRFSGSGS
GTDFIFTISSLQPEDIATYYCLQSFNGPLTFGGGTKVEIKEQ
KLISEEDL
SEQ ID NO: 34 {#13}
Length: 499 Type: protein Organism: human Sequence:
MALPVTALLLPLALLLHAARPQVQLQESGGGLVKPGGSLKL
SCAASGFTFSKFGMSWVRQTPDKRLEWVASISTGGYNTFY
SDNVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARGYS
STSFAMDYWGQGTMVTVSSGSTSGSGKPGSGEGSDIQMTQ
SPSSLSASVGDRVTITCMTSTDIDDDMNWYQQKPGKTPKLL
IYEGNTLRPGVPSRFSGSGSGTDFIFTISSLQPEDIATYYCL
QSFNVPLTFGGGTKVEIKEQKLISEEDLGGGPRKPTTTPAP
RPPTPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFALAYLL
DGILFIYGVILTALFLRVKRGRKKLLYIFKQPFMRPVQTTQEE
DGCSCRFPEEEEGGCELKFSRSADAPAYQQGQNQLYNELN
LGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQK
DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL
HMQALPPR
Date Recue/Date Received 2021-03-05 32 SEQ ID NO: 35 {#14}
Length: 499 Type: protein Organism: human Sequence:
MALPVTALLLPLALLLHAARPQVQLQESGGGLVKPGGSLKL
SCAASGFTFSKFGMSWVRQTPDKRLEWVASIDPENDETFY
SDNVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARGYG
STSFAMDYWGQGTMVTVSSGSTSGSGKPGSGEGSDIQMTQ
SPSSLSASVGDRVTITCMTSTDIDDDMNWYQQKPGKTPKLL
IYEGNTLRPGVPSRFSGSGSGTDFIFTISSLQPEDIATYYCL
QSFNGPLTFGGGTKVEIKEQKLISEEDLGGGPRKPTTTPAP
RPPTPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFALAYLL
DGILFIYGVILTALFLRVKRGRKKLLYIFKQPFMRPVQTTQEE
DGCSCRFPEEEEGGCELKFSRSADAPAYQQGQNQLYNELN
LGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQK
DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL
HMQALPPR
SEQ ID NO: 36 {#15}
Length: 349 Type: protein Organism: human Sequence:
MGVKVLFALICIAVAEAVSDVPRDLEVVAATPTSLLISWDAP
AVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGV
DYTITVYAVTPRGDWNEGSKPISINYRTEQKLISEEDLGGGP
RKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR
GLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPF
MRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY
QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRR
KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ
GLSTATKDTYDALHMQALPPR
SEQ ID NO: 37 {#16}
Length: 353 Type: protein Organism: human Sequence:
MALPVTALLLPLALLLHAARPVSDVPRDLEVVAATPTSLLIS
WDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGL
KPGVDYTITVYAVTPRGDWNEGSKPISINYRTEQKLISEEDL
GGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA
VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIF
Date Re9ue/Date Received 2021-03-05 33 KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAD
APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP
QRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG
LYQGLSTATKDTYDALHMQALPPR
SEQ ID NO: 38 {YYB 107}
Length: 351 Type: protein Organism: human Sequence:
MGWSCIILFLVATATGVHSVSDVPRDLEVVAATPTSLLISWD
APAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKP
GVDYTITVYAVTPRGDWNEGSKPISINYRTEQKLISEEDLGG
GPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH
TRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQ
PFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP
AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQ
RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL
YQGLSTATKDTYDALHMQALPPR
SEQ ID NO: 39 {YYB 105}
Length: 497 Type: protein Organism: human Sequence:
MGWSCIILFLVATATGVHSQVQLQESGGGLVKPGGSLKLSC
AASGFTFSKFGMSWVRQTPDKRLEWVASISTGGYNTFYSD
NVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARGYSST
SFAMDYWGQGTMVTVSSGSTSGSGKPGSGEGSDIQMTQSP
SSLSASVGDRVTITCMTSTDIDDDMNWYQQKPGKTPKLLIY
EGNTLRPGVPSRFSGSGSGTDFIFTISSLQPEDIATYYCLQS
FNVPLTFGGGTKVEIKEQKLISEEDLGGGPRKPTTTPAPRPP
TPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFALAYLLDGI
LFIYGVILTALFLRVKRGRKKLLYIFKQPFMRPVQTTQEEDG
CSCRFPEEEEGGCELKFSRSADAPAYQQGQNQLYNELNLG
RREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK
MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM
QALPPR
SEQ ID NO: 40 {T2A peptide}
Length: 21 Type: protein Organism: human Sequence:
Date Recue/Date Received 2021-03-05 34 GSGEGRGSLLTCGDVEENPGP
SEQ ID NO: 41 {P2A peptide}
Length: 22 Type: protein Organism: human Sequence:
GSGATNFSLLKQAGDVEENPGP
SEQ ID NO: 42 {E2A peptide}
Length: 23 Type: protein Organism: human Sequence:
GSGQCTNYALLKLAGDVESNPGP
SEQ ID NO: 43 {F2A peptide}
Length: 25 Type: protein Organism: human Sequence:
GSGVKQTLNFDLLKLAGDVESNPGP
Date Recue/Date Received 2021-03-05 35
As a result of the analysis, as shown in FIG. 17, the expression of the chimeric switch receptors of TGF8R2 and IL-18R in the 4 groups (#1, #2, #7, and #8) after 24 hours of co-culture showed a tendency of a decrease in the groups co-cultured with glioma cell line, U87 cells compared to those cultured with CAR-T cells only, but the average expression rate was 30% (FIG. 17). The expression of the chimeric switch receptors of TGF8R2 and IL-18R in the 4 groups (#1, #2, #7, and #8) after 48 hours of co-culture showed a tendency of a decrease in the groups co-cultured with U87 cells compared to those cultured with CAR-T cells only, but the average expression rate was 25%. The expression of the chimeric switch receptors of TGF8R2 and IL-18R in the 4 groups (#1, #2, #7, and #8) after 72 hours of co-culture showed a tendency of a decrease in the groups co-cultured with U87 cells compared to those cultured with CAR-T cells only, but the average expression rate was 15%. Although the changes in the expression rate were greater compared to one without co-culture with target cells or the co-culture with 293FT cells (which were used as the control group for normal cells), by the confirmation of the results of the chimeric switch receptor expression rate of TGF8R2 and IL-18R against glioma cell line in which IL13Ra2 is overexpressed, the chimeric antigen receptor (CAR) according to the present invention is expected to show excellent in vivo persistence and anti-tumor effects of CAR-T cells in a hostile tumor microenvironment.
Experimental Example 8: Checking of cytokine (IFN-v) production by T cells, which were transformed with novel chimeric antigen receptors, against target cells Experimental Methods In order to confirm the cytokine (IFN-y) production after 24 hours and 48 hours of co-culture between the CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12) and glioma cell line (U87 cells) and 293FT target cells (which were used as a control group for normal cells), the CAR-T cells (effector cells) in which the CAR
expression rate is in the range of 20% to 40% were used 10 days after activation of the cells with an anti-CD3 Ab. The CAR-T cells were added to a 6-well plate at a ratio in which effector: target was 1x106 cells: 2x106 cells, 6 mL of a culture medium was added per well, and the cells reacted at 37 C for 24 hours. 100 pL of the supernatant was collected and transferred to a 1.5 mL tube, the cells were cultured further up to 48 hours, and 100 pL of the supernatant was collected from the culture in the same manner.
The IFN-y analysis experiment was performed as follows according to the manufacturer's instructions of the ELISA analyzer (R&D systems). 3 mL of the Date Recue/Date Received 2021-03-05 18 calibrator diluent RD6-21 was added to an IFN-y standard bottle and mixed in a shaker for 15 minutes, dispensed in an amount of 1 mL per 1.5 mL tube to prepare two "standard rs. 500 pL was taken from 1 mL of the "standard 1" dispensed and was subjected to serial dilution to prepare up to "standard 7". In order to prepare blanks, 500 pL of the culture medium and 500 pL of the calibrator diluent RD6-21 were mixed.
In order to dilute the samples by 1/20, 190 pL of the calibrator diluent RD6-was added into each of new 1.5 mL tubes as many as the number of samples. The assay samples were each prepared to a total volume of 200 pL by collecting 10 pL of the supernatant of each sample. To prepare a wash buffer, 500 mL of distilled water and 20 mL of wash buffer concentrate were mixed well in a 500 mL storage bottle.
Assay diluent RD1-51 (blue dye) in an amount of 100 pL each was added to the IFN-y microplate for each well of "standard 1" to "standard 7", blank, and samples. After adding 100 pL each of the blank, standards, and samples prepared above, a plate sealer was attached thereto and reacted at room temperature for 2 hours. After the reaction, the reaction solution was discarded, and 400 pL of the prepared wash buffer was added thereto, and the wells were washed 4 times. 200 pL of IFN-y conjugate was added to each well, a plate sealer was attached thereto, and the reaction was performed at room temperature for 2 hours. The reaction solution was discarded, and the wells were washed 4 times using 400 pL of the wash buffer. After mixing the color reagent A: B in a 1:1 ratio, the mixture was added in an amount of 200 pL per well, a plate sealer was attached thereto, the light was blocked with foil, and the reaction was performed at room temperature for 30 minutes. After the reaction, 50 pL of the stop solution was added to each well and measured at 450 nm within 30 minutes.
Experimental Results YYB103 was shown to secrete a relatively high amount of IFN-y compared to other samples. When CAR-T cells were cultured in the presence of U87 cells, CAR-T
(#1, #2, #5, and #6) containing an IL-7Ra signaling domain were shown to contain more IFN-y compared to CAR-T (#7, #8, #11, and #12) containing an IL-21R8 signaling domain. Results between samples after 24 hours and 48 hours of incubation showed similar patterns (FIG. 18).
As shown in FIG. 12, when the cytotoxicity was confirmed through LDH assay after 24 hours of co-culture between the CAR-T (which were transformed with the chimeric antigen receptor (CAR) of the present invention), U87 cells (human glioma cell line), and 293FT target cells (which are used as a control group for normal cells), the 8 groups showed similar abilities of killing target cells. However, considering that the production of IFN-y was small and thus target cells could be killed without adverse effects caused by cytokines (cytokine release syndrome, CRS), in terms of safety, the effect of the CAR-T cell therapeutic is expected to have a fewer side effects while having excellent therapeutic effects.
Experimental Example 9: Checking of cytokine (IL-21) production by CAR-T
cells, which were transformed with novel chimeric antigen receptors Experimental Methods In order to confirm the cytokine (IL-21) production after 24 hours and 48 hours of co-culture between the CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, Date Recue/Date Received 2021-03-05 19 or #12) and glioma U87 cells and 293FT target cells (which were used as a control group for normal cells), the CAR-T cells (effector cells) in which the CAR
expression rate is in the range of 20% to 40% were used 10 days after activation of the cells with an anti-CD3 Ab. The CAR-T cells were added to a 6-well plate at a ratio in which effector: target was 1x106 cells: 2x106 cells, 6 mL of a culture medium was added per well, and the cells were reacted at 37 C for 24 hours. Then, 100 pL of the supernatant was collected and transferred to a 1.5 mL tube, the cells were cultured further up to 48 hours, and 100 pL of the supernatant was collected from the culture in the same manner.
The IL-21 analysis experiment was performed as follows according to the manufacturer's instructions of the ELISA analyzer (Invitrogen). 100 pL of a capture antibody was added to each well of a 96-well ELISA plate and reacted at 4 C
overnight.
After the reaction, the wells were washed 3 times using 250 pL of wash buffer.
ELISA/ELISASPOT diluent was added in an amount of 200 pL per well and reacted at room temperature for one hour.
After the reaction, 100 pL each of the prepared blank, cytokine IL-21 standard, and samples were added thereto, and ELISA/ELISASPOT diluent was added in an amount of 100 pL per well, and a plate sealer was attached thereto and the reaction was performed at room temperature for two hours. After the reaction, the wells were washed 3 times using 250 pL of wash buffer. 100 pL of a detection antibody was added to each well, and a plate sealer was attached thereto and the reaction was performed at room temperature for one hour. After the reaction, the wells were washed 3 times using 250 pL of wash buffer. 100 pL of Avidin-HRP was added to each well, a plate sealer was attached thereto, and the reaction was performed at room temperature for minutes. After the reaction, the wells were washed 5 times using 250 pL of wash buffer.
100 pL of lx TMB solution was added to each well, a plate sealer was attached thereto, and the reaction was performed at room temperature for 30 minutes. After the reaction, 50 pL of the stop solution was added to each well and measured at 450 nm within 30 minutes.
Experimental Results It was confirmed that IL-21 was secreted in IL-21 gene-containing CAR-T cells (#1, #5, #7, and #11). When the CAR-T cells were co-cultured with U87 cells, a greater amount of IL-21 was secreted, and considering that the overall experimental results at 24 hours and 48 hours were similar, no significant difference according to the incubation time was observed. As the result of the experiment at 48 hours showed a higher concentration than that at 24 hours, the secretion of IL-21 appeared to proceed steadily (FIG. 19).
The secretion of IL-21 in an environment of cancer cells is speculated to increase the ability to kill cancer cells by assisting the activation of innate immune-related cells.
Example 3: Preparation of cancer antigen EGFRvIll targeting CAR vector and aV3 targeting CAR vector In the case of EGFRvIll, which is a major antigen for tumors such as glioblastoma and lung cancer, in order to reduce the side effects of CAR-T
cells through non-specific binding while minimizing the binding affinity for EGFR wild-type in which Date Recue/Date Received 2021-03-05 20 the specificity for EGFRvIll is maintained, in SEQ ID NO: 33, positions 52 to 57 of SEQ
ID NO: 3 were first changed from STGGYN to DPENDE (a CDR2 part of a heavy chain), position 101 of SEQ ID NO: 3 was changed from S to G (a CDR3 part of a heavy chain), and position 229 of SEQ ID NO: 3 was changed from V to G (a CDR3 part of a light chain).
Human CD3 (P20963-1), human CD8A (P01732), human 4-1BB (Q07011), human CD3Z (P20963), a Gaussia princeps luciferase, and human kappa light chain signal sequence (HuVHCAMP) were optimized using scientific literature and publicly available databases, and thereby chimeric antigen receptor-containing polypeptides consisting of codon-optimized synthetic DNA (#13, #14, #15, and #16 of SEQ ID
NOS: 34 to 37) were prepared.
The completed structure of CAR-containing polypeptide #13 includes a Kozak consensus ribosome-binding sequence, a CD8A signal sequence, an antigen binding domain which binds to EGFRvIll of SEQ ID NO: 3, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8A, a human 0D8 transmembrane domain, a cytoplasmic 4-1BB costimulatory signal domain, a CD3 cytoplasmic domain (SEQ ID NO: 18), and an Xhol/Notl cleavage site.
The finally prepared CAR gene fragment was conjugated to the MFG retrovirus expression vector digested with Xhol/Notl (Emtage PC et al., Clin Cancer Res, 2008, 14:8112-8122). In this Example, in order to compare the activity of chimeric antigen receptors, YYB105 (SEQ ID NO: 39; see PCT International Publication No. WO 2017/023138) was additionally prepared.
The completed structure of CAR-containing polypeptide #14 includes a Kozak consensus ribosome-binding sequence, a CD8A signal sequence, an antigen binding domain which binds to EGFRvIll of SEQ ID NO: 32, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8A, a human 0D8 transmembrane domain, a cytoplasmic 4-1BB costimulatory signal domain, a CD3 cytoplasmic domain (SEQ ID NO: 18), and an Xhol/Notl cleavage site. The finally prepared CAR gene fragment was conjugated to the MFG retrovirus expression vector digested with Xhol/Notl (Emtage PC et al., Clin Cancer Res, 2008,14:8112-8122).
In the case of aV8 anti-angiogenic, the CAR expression rate and persistence in CAR-T cells targeting these were optimized. In the case of aVp, the CAR
expression rate and persistence in CAR-T cells targeting these were optimized using Gaussia princeps luciferase signal sequence or 0D8 signal sequence (SEQ ID NO: 36 and SEQ
ID NO: 37).
The completed structure of CAR-containing polypeptide #15 includes a Kozak consensus ribosome-binding sequence, a Gaussia princeps luciferase signal sequence, SEQ ID NO: 5 (an antigen binding domain which binds to aV8), three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a Date Recue/Date Received 2021-03-05 21 CAR protein, a hinge region of human CD8A, a human 0D8 transmembrane domain, a cytoplasmic 4-1BB costimulatory signal domain, a CD3 cytoplasmic domain (SEQ
ID
NO: 18), and an Xhol/Notl cleavage site. The finally prepared CAR gene fragment was conjugated to the MFG retrovirus expression vector digested with Xhol/Notl (Emtage PC etal., Clin Cancer Res, 2008, 14:8112-8122). In this Example, in order to compare the activity of chimeric antigen receptors, YYB107 (SEQ ID NO: 38; see PCT
International Publication No. WO 2017/023138) was additionally prepared.
The completed structure of CAR-containing polypeptide #16 includes a Kozak consensus ribosome-binding sequence, a Gaussia princeps luciferase signal sequence, SEQ ID NO: 5 (an antigen binding domain which binds to aV8), three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8A, a human 0D8 transmembrane domain, a cytoplasmic 4-1BB costimulatory signal domain, a CD3 cytoplasmic domain (SEQ
ID
NO: 18), and an Xhol/Notl cleavage site. The finally prepared CAR gene fragment was conjugated to the MFG retrovirus expression vector digested with Xhol/Notl (Emtage PC etal., Clin Cancer Res, 2008, 14:8112-8122).
Example 4: Preparation of CAR-T cells transformed with chimeric antigen receptors High-titer CAR-expressing PG13 clones were prepared such that Phoenix-Ampho and Phoenix-Eco cells were transiently transfected with the retroviral expression vector prepared in Example 1, and then, cell-free vector stocks were prepared from the transfected Phoenix-Ampho and Phoenix-Eco cells by transfecting PG13 cells.
For high-titer monoclones, PG13/#13, PG13/#14, PG13/#15, and PG13/#16 cells were stained using an anti-myc Ab (BD Pharmingen), and these monoclones were isolated using a flow cytometer. The transduction degree of PG13/#13, PG13/#14, PG13/#15, and PG13/#16 cells, which were transduced using an anti-myc Ab, was analyzed using a flow cytometer. The supernatants of the transduced PG13/#13, PG13/#14, PG13/#15, and PG13/#16 cells contained retrovirus, and the supernatants were collected for genetic modification of T cells. The peripheral blood mononuclear cells (PBMCs) were separated using centrifugation by adding the whole blood obtained from a healthy human donor into Ficoll Paque (GE Healthcare). The separated PBMCs were cultured by adding an anti-CD3 monoclonal antibody (eBioscience) at a concentration of 100 ng/mL under the condition of human IL-2 (Novartis) at a concentration of 100 IU/mL
to activate the T cell fraction (BL Levine, Cancer Gene Therapy, 2015, 22:79-84). Two to three days after the cultivation, most of the cells were T cells and included natural killer cells at a percentage of 0% to 2%. Two to three days after the activation step, the T cells were subjected to transduction two times over two days using the retroviral supernatant and washed, and then proliferated for 14 days in a flask. IL-2 was maintained at a concentration of 100 IU/mL. The T cells modified as such were used for an analysis experiment.
Experimental Example 1: Checking of growth rate and viability of CAR-T cells transformed with chimeric antigen receptors Experimental Results Date Recue/Date Received 2021-03-05 22 For the T cells prepared in Example 4 above, the number of cells was counted to confirm the growth rate and viability rate of CAR-T, and the results are shown in FIG. 20.
The number of cells and growth rate of all of the groups (#13, #14, #15, and #16) were shown to be similar to those of the control group (i.e., YYB105) depending on the day of the week, the cells were shown to grow rapidly from Day 12 of culture, and the viability was also shown to be 90% or higher (see FIG. 20).
Experimental Example 2: Checking of CAR expression rate on the cell surface of CAR-T cells transformed with chimeric antigen receptors Experimental Methods (flow cytometric analysis) For flow cytometry (>30,000 events), a BD LSRII device (Becton Dickinson) and BD FACSDiva software (Becton Dickinson) were used. Specifically, the cells were washed once with PBS containing 2% bovine serum albumin before adding a PE-conjugated anti-myc antibody (BD Pharmingen) thereto. After washing, the cells were reacted with each antibody at 4 C for 30 minutes in a state where light was blocked and washed once, and thereafter, the expression rate of CAR on the surface of transduced T cells was checked.
Experimental Results In order to confirm whether the 4 kinds of CAR prepared in Example 2 (#13, #14, #15, and #16) were expressed on the T cell surface, T cell cultivation was performed for 14 days according to Example 4, and then flow cytometric analysis was performed according to the experimental method.
As a result of the analysis, as shown in FIG. 21, the expression rate of the chimeric antigen receptors expressed on the surface of live T cells was shown to increase in #13 (43.0%) and #14 (50.8%) compared to YYB105 (37.4%) (i.e., a control group) (FIG. 21); and was shown to increase in #15 (45.9%) and #16 (40.0%) compared to YYB107 (24.6%) (i.e., a control group) (FIG. 21).
[Industrial Applicability]
The present invention relates to rapidly developing a CAR-T cell in the field of cancer treatment. The CAR-T cell according to the present invention has a remarkably excellent expression rate and persistence, and thus has an improved therapeutic effect for solid cancer, etc., and can be effectively used in the field of customized cancer treatment.
[SEQUENCE LISTING]
SEQ ID NO: 1 {antigen binding wild type IL13 domain binding to IL13Ra2}
Length: 112 Type: ligand protein Organism: human Sequence:
GPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAG
MYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSL
HVRDTKIEVAQFVKDLLLHLKKLFREGQFN
Date Recue/Date Received 2021-03-05 23 SEQ ID NO: 2 {antigen binding domain capable of binding to an antigen associated with an angiogenic activity}
Length: 92 Type: ligand protein Organism: human Sequence:
EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFT
VLQPPSTATISGLKPGVDYTITVYAVVERNGRELNTPPISINY
RTHHHHHH
SEQ ID NO: 3 {antigen binding domain binding to EGFRvIll}
Length: 252 Type: scFv protein Organism: human Sequence:
QVQLQESGGGLVKPGGSLKLSCAASGFTFSKFGMSWVRQT
PDKRLEWVASISTGGYNTFYSDNVKGRFTISRDNAKNTLYL
QMSSLKSEDTAMYYCARGYSSTSFAMDYWGQGTMVTVSSG
STSGSGKPGSGEGSDIQMTQSPSSLSASVGDRVTITCMTST
DIDDDMNWYQQKPGKTPKLLIYEGNTLRPGVPSRFSGSGSG
TDFIFTISSLQPEDIATYYCLQSFNVPLTFGGGTKVEIKEQKL
ISEEDL
SEQ ID NO: 4 {antigen binding domain binding to EphA2}
Length: 141 Type: ligand protein Organism: human Sequence:
DRHTVFWNSSNPKFRNEDYTIHVQLNDYVDIICPHYEDHSV
ADAAMEQYILYLVEHEEYQLCQPQSKDQVRWQCNRPSAKH
GPEKLSEKFQRFTAFALAKEFKAGHSYYYISKPIHQHEDRCL
RLKVTVSGEQKLISEEDL
SEQ ID NO: 5 {antigen binding domain binding to aVp}
Length: 104 Type: ligand protein Organism: human Sequence:
VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGG
NSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTPRGDWN
EGSKPISINYRTEQKLISEEDL
SEQ ID NO: 6 {antigen binding domain binding to glypican1}
Date Recue/Date Received 2021-03-05 24 Length: 418 Type: ligand protein Organism: human Sequence:
TSPCDNFDCQNGAQCIVRINEPICQCLPGYQGEKCEKLVSV
NFINKESYLQIPSAKVRPQTNITLQIATDEDSGILLYKGDKDH
lAVELYRGRVRASYDTGSHPASAIYSVETINDGNFHIVELLA
LDQSLSLSVDGGNPKIITNLSKQSTLNFDSPLYVGGMPGKS
NVASLRQAPGQNGTSFHGCIRNLYINSELQDFQKVPMQTGI
LPGCEPCHKKVCAHGTCQPSSQAGFTCECQEGWMGPLCD
QRTNDPCLGNKCVHGTCLPINAFSYSCKCLEGHGGVLCDEE
EDLFNPCQAIKCKHGKCRLSGLGQPYCECSSGYTGDSCDR
EISCRGERIRDYYQKQQGYAACQTTKKVSRLECRGGCAGG
QCCGPLRSKRRKYSFECTDGSSFVDEVEKVVKCGCTRCVS
EQKLISEEDL
SEQ ID NO: 7 {antigen binding domain binding to mesothelin}
Length: 262 Type: scFv protein Organism: human Sequence:
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSG
STNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGKNGAFDIWGQGTMV
TVSSGSTSGSGKPGSGEGSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWN
WI RQSPS RGLEWLGRTYYRSKVVYN DYAVSVKSRMS IN PDTSKNQFSLQLNSVTP
EDTAVYYCARGMMTYYYGMDV WGQGTTVTVSSGILGS
SEQ ID NO: 8 {hinge region sequence -1}
Length: 47 Type: protein Organism: human Sequence:
KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG
LDFACD
SEQ ID NO: 9 {hinge region sequence -2}
Length: 45 Type: protein Organism: human Sequence:
KPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVHTRG
LDFA
SEQ ID NO: 10 {transmembrane domain sequence-1}
Date Recue/Date Received 2021-03-05 25 Length: 21 Type: protein Organism: human Sequence:
IYIWAPLAGTCGVLLLSLVIT
SEQ ID NO: 11 {transmembrane domain sequence-2}
Length: 23 Type: protein Organism: human Sequence:
LAYLLDGILFIYGVILTALFLRV
SEQ ID NO: 12 {4-1BB}
Length: 42 Type: protein Organism: human Sequence:
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE
L
SEQ ID NO: 13 {Wild type CD28}
Length: 41 Type: protein Organism: human Sequence:
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYR
S
SEQ ID NO: 14 {IL-7Ra}
Length: 459 Type: protein Organism: human Sequence:
MTILGTTFGMVFSLLQVVSGESGYAQNGDLEDAELDDYSFSCYSQLEVNGSQHSLTC
AFEDPDVNITNLEFEICGALVEVKCLNFRKLQEIYFIETKKFLLIGKSNICVKVGEKSLTCK
KIDLTTIVKPEAPFDLSVVYREGANDFVVTFNTSHLQKKYVKVLMHDVAYRQEKDENK
VVTHVNLSSTKLTLLQRKLQPAAMYEIKVRSIPDHYFKGFWSEWSPSYYFRTPEINNSS
GEMDPILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVS
FNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV
VITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNS
TLPPPFSLQSGILTLNPV AQGQPILTSLGSNQEEAYVTMSSFYQNQ
SEQ ID NO: 15 {part of IL-7Ra}
Date Recue/Date Received 2021-03-05 26 Length: 64 Type: protein Organism: human Sequence:
KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGF
LQDTF
SEQ ID NO: 16 {IL-2R8}
Length: 551 Type: protein Organism: human Sequence:
MAAPALSWR LP LLI LLLPLATSWASAAVNGTSQFTCFYNS RAN ISCVWSQDGALQDTS
CQVHAWPDRRRWNQTCELLPVSQASWACN LI LGAPDSQKLTTVDIVTLRVLCREGVR
WRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSP
GHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRT
KPAALGKDTI PWLGH LLVGLSGAFGFI I LVYLLI NCRNTGPWLKKVLKCNTPDPSKFFSQ
LSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASL
SSNHSLTSCFTNQGYFFFHLPDALEI EACQVYFTYDPYSE EDPDEGVAGAPTGSS PQP
LQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVP
RDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQG
EFRALNARLPLNTD AYLSLQELQGQDPTHLV
SEQ ID NO: 17 {part of IL-2R8}
Length: 104 Type: protein Organism: human Sequence:
NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEI
SPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHL
SEQ ID NO: 18 {CD3}
Length: 113 Type: protein Organism: human Sequence:
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG
RDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGE
RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
SEQ ID NO: 19 {TGF-8R2 exodomain}
Length: 137 Type: protein Organism: human Date Recue/Date Received 2021-03-05 27 Sequence:
TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKP
QEVCVAVWRKNDENITLETVCHDPKLPYHDFI LEDAASPKCI MKEKKKPGETFFMCSC
SSDECNDNIIFSEEYNTSNPD
SEQ ID NO: 20 {transmembrane domain and endodomain of IL-18R}
Length: 219 Type: protein Organism: human Sequence:
ECRPENGEEHTFAVEILPRVLEKHFGYKLCIFERDVVPGGAVVDEIHSLIEKSRRLIIVLS
KSYMSNEVRYELESGLHEALVERKI KI I LI EFTPVTDFTFL
PQSLKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPV LSES
SEQ ID NO: 21 {IL-21}
Length: 133 Type: protein Organism: human Sequence:
QGQDRH MI RM RQLI DIVDQLKNYVN DLVPE FLPAPE DVETNCEWSAFSCFQKAQLKSA
NTGNNERI I NVSI KKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK
EFLERFKSLLQKMIHQHLSSRTHGSEDS
SEQ ID NO: 22 {YYB 103}
Length: 359 Type: protein Organism: human Sequence:
MGWSCIILFLVATATGVHSGPVPPSTALRKLIEELVNITQNQ
KAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQD
MLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKL
FKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEAC
RPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRG
RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRV
KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD
PEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR
RGKGHDGLYQGLSTATKDTYDALHMQALPPR
SEQ ID NO: 23 {#1}
Length: 998 Type: protein Organism: human Sequence:
Date Recue/Date Received 2021-03-05 28 MGWSCI I LFLVATATGVHSGPVPPSTALRKLI EE LVN ITQNQKAPLCNGS MVWS IN LTA
GMYCAALESLINVSGCSAIEKTQDMLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLL
LHLKKLFKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR
GLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYI FKQPFM RPVQTTQE EDGCSCR
FPEEEEGGCELKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVD
DIQARDEVEGFLQDTFRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR
DPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYLPQSTA
TKDTYDYVTMQALPPREGRGS LLTCGDVEEN PGPMALPVTALLLPLALLLHAARPTI PP
HVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEV
CVAVWRKNDE NITLETVCHDPKLPYHDFI LE DAASPKCI MKEKKKPGETFFMCSCSSD
DETLTDGKTYDAFVSYLKECRPENGEEHTFAVEI LPRVLEKHFGYKLCI FE RDVVPGGA
VVDE I HSLI EKSRRLI IVLSKSYMSNEVRYELESGLHEALVERKIKI I LI EFTPVTDFTFLPQ
SLKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPVLSESRRKR
SGSGATNFSLLKQAGDVEE NPGPMYRMQLLSCIALSLALVTNSQGQDRH MI RMRQLI D
IVDQLKNYVN DLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGN N ERI I NVSI KKL
KRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSE
DS
SEQ ID NO: 24 {#2}
Length: 818 Type: protein Organism: human Sequence:
MGWSCI I LFLVATATGVHSGPVPPSTALRKLI EE LVN ITQNQKAPLCNGS MVWS IN LTA
GMYCAALESLINVSGCSAIEKTQDMLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLL
LHLKKLFKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR
GLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYI FKQPFM RPVQTTQE EDGCSCR
FPEEEEGGCELKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVD
DIQARDEVEGFLQDTFRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR
DPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYLPQSTA
TKDTYDYVTMQALPPREGRGS LLTCGDVEEN PGPMALPVTALLLPLALLLHAARPTI PP
HVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEV
CVAVWRKNDE NITLETVCHDPKLPYHDFI LE DAASPKCI MKEKKKPGETFFMCSCSSD
DETLTDGKTYDAFVSYLKECRPENGEEHTFAVEI LPRVLEKHFGYKLCI FE RDVVPGGA
VVDE I HSLI EKSRRLI IVLSKSYMSNEVRYELESGLHEALVERKIKI I LI EFTPVTDFTFLPQ
SLKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKT VKPGRDEPEVLPVLSES
SEQ ID NO: 25 {#5}
Length: 594 Type: protein Organism: human Sequence:
Date Recue/Date Received 2021-03-05 29 MGWSCI I LFLVATATGVHSGPVPPSTALRKLI EE LVN ITQNQKAPLCNGS MVWS IN LTA
GMYCAALESLINVSGCSAIEKTQDMLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLL
LHLKKLFKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR
GLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYI FKQPFM RPVQTTQE EDGCSCR
FPEEEEGGCELKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVD
DIQARDEVEGFLQDTFRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR
DPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYLPQSTA
TKDTYDYVTMQALPPREGRGSLLTCGDVEENPGPMYRMQLLSCIALSLALVTNSQGQ
DRH MI RM RQLI DIVDQLKNYVN DLVPEFLPAPE DVETNCEWSAFSCFQKAQLKSANTG
NNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIH
QH LSSRTHGSEDS
SEQ ID NO: 26 {#6}
Length: 423 Type: protein Organism: human Sequence:
MGWSCI I LFLVATATGVHSGPVPPSTALRKLI EE LVN ITQNQKAPLCNGS MVWS IN LTA
GMYCAALESLINVSGCSAIEKTQDMLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLL
LHLKKLFKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR
GLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYI FKQPFM RPVQTTQE EDGCSCR
FPEEEEGGCELKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVD
DIQARDEVEGFLQDTFRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR
DPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYLPQSTA
TKDTYDYVTMQALPPR
SEQ ID NO: 27 {#7}
Length: 1038 Type: protein Organism: human Sequence:
KMIHQHLSSRTHGSEDS
SEQ ID NO: 28 {#8}
Length: 858 Type: protein Organism: human Sequence:
MGWSCI I LFLVATATGVHSGPVPPSTALRKLI EE LVN ITQNQKAPLCNGS MVWS IN LTA
GMYCAALESLINVSGCSAIEKTQDMLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLL
LHLKKLFKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR
GLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYI FKQPFM RPVQTTQE EDGCSCR
FPEEEEGGCELNCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPS
SSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHL
Date Recue/Date Received 2021-03-05 30 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNP
QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYLSLSTATKDTYLPQHMQALPP
REGRGSLLTCGDVE EN PGPMALPVTALLLPLALLLHAARPTI PPHVQKSVN N DM IVTDN
NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLET
VCHDPKLPYHDFI LE DAASPKCI MKEKKKPGETFFMCSCSSDECNDNI I FSE EYNTSNP
DPGHVFTRG M I IAVLI LVAVVCLVTVCVIYRVDLVLFYR H LTR R DETLTDGKTYDAFVSY
LKECRPENGEEHTFAVEI LPRVLEKHFGYKLCI FERDVVPGGAVVDEI HSLI EKSRRLI IV
LSKSYMS N EVRYELESGLH EALVE RKI KI I LI EFTPVTDFTFLPQSLKLLKSH RVLKWKAD
KSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPVLSES
SEQ ID NO: 29 {#11}
Length: 634 Type: protein Organism: human Sequence:
MGWSCI I LFLVATATGVHSGPVPPSTALRKLI EE LVN ITQNQKAPLCNGS MVWS IN LTA
GMYCAALESLINVSGCSAIEKTQDMLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLL
LHLKKLFKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR
GLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYI FKQPFM RPVQTTQE EDGCSCR
FPEEEEGGCELNCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPS
SSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHL
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNP
QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYLSLSTATKDTYLPQHMQALPP
REGRGSLLTCGDVE ENPGPMYRMQLLSCIALSLALVTNSQGQDRH MI RMRQLI DIVDQ
LKNYVN DLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGN N ERI I NVS I KKLKRKP
PSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS
SEQ ID NO: 30 {#12}
Length: 463 Type: protein Organism: human Sequence:
MGWSCI I LFLVATATGVHSGPVPPSTALRKLI EE LVN ITQNQKAPLCNGS MVWS IN LTA
GMYCAALESLINVSGCSAIEKTQDMLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLL
LHLKKLFKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR
GLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYI FKQPFM RPVQTTQE EDGCSCR
FPEEEEGGCELNCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPS
SSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHL
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNP
QEGLYNELQKDKMAEAYSEI
GMKGERRRGKGHDGLYLSLSTATKDTYLPQHMQALPPR
SEQ ID NO: 31 {mutated 3rd ITAM-1}
Length: 23 Date Recue/Date Received 2021-03-05 31 Type: protein Organism: human Sequence:
YLPQSTATKDTYDYVTMQALPPR
SEQ ID NO: 32 {mutated 3rd ITAM-2}
Length: 23 Type: protein Organism: human Sequence:
YLSLSTATKDTYLPQHMQALPPR
SEQ ID NO: 33 {antigen binding domain binding to EGFRvIll}
Length: 252 Type: scFv protein Organism: human Sequence:
QVQLQESGGGLVKPGGSLKLSCAASGFTFSKFGMSWVRQT
PDKRLEWVASIDPENDETFYSDNVKGRFTISRDNAKNTLYL
QMSSLKSEDTAMYYCARGYGSTSFAMDYWGQGTMVTVSS
GSTSGSGKPGSGEGSDIQMTQSPSSLSASVGDRVTITCMTS
TDIDDDMNWYQQKPGKTPKLLIYEGNTLRPGVPSRFSGSGS
GTDFIFTISSLQPEDIATYYCLQSFNGPLTFGGGTKVEIKEQ
KLISEEDL
SEQ ID NO: 34 {#13}
Length: 499 Type: protein Organism: human Sequence:
MALPVTALLLPLALLLHAARPQVQLQESGGGLVKPGGSLKL
SCAASGFTFSKFGMSWVRQTPDKRLEWVASISTGGYNTFY
SDNVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARGYS
STSFAMDYWGQGTMVTVSSGSTSGSGKPGSGEGSDIQMTQ
SPSSLSASVGDRVTITCMTSTDIDDDMNWYQQKPGKTPKLL
IYEGNTLRPGVPSRFSGSGSGTDFIFTISSLQPEDIATYYCL
QSFNVPLTFGGGTKVEIKEQKLISEEDLGGGPRKPTTTPAP
RPPTPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFALAYLL
DGILFIYGVILTALFLRVKRGRKKLLYIFKQPFMRPVQTTQEE
DGCSCRFPEEEEGGCELKFSRSADAPAYQQGQNQLYNELN
LGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQK
DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL
HMQALPPR
Date Recue/Date Received 2021-03-05 32 SEQ ID NO: 35 {#14}
Length: 499 Type: protein Organism: human Sequence:
MALPVTALLLPLALLLHAARPQVQLQESGGGLVKPGGSLKL
SCAASGFTFSKFGMSWVRQTPDKRLEWVASIDPENDETFY
SDNVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARGYG
STSFAMDYWGQGTMVTVSSGSTSGSGKPGSGEGSDIQMTQ
SPSSLSASVGDRVTITCMTSTDIDDDMNWYQQKPGKTPKLL
IYEGNTLRPGVPSRFSGSGSGTDFIFTISSLQPEDIATYYCL
QSFNGPLTFGGGTKVEIKEQKLISEEDLGGGPRKPTTTPAP
RPPTPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFALAYLL
DGILFIYGVILTALFLRVKRGRKKLLYIFKQPFMRPVQTTQEE
DGCSCRFPEEEEGGCELKFSRSADAPAYQQGQNQLYNELN
LGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQK
DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL
HMQALPPR
SEQ ID NO: 36 {#15}
Length: 349 Type: protein Organism: human Sequence:
MGVKVLFALICIAVAEAVSDVPRDLEVVAATPTSLLISWDAP
AVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGV
DYTITVYAVTPRGDWNEGSKPISINYRTEQKLISEEDLGGGP
RKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR
GLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPF
MRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAY
QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRR
KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ
GLSTATKDTYDALHMQALPPR
SEQ ID NO: 37 {#16}
Length: 353 Type: protein Organism: human Sequence:
MALPVTALLLPLALLLHAARPVSDVPRDLEVVAATPTSLLIS
WDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGL
KPGVDYTITVYAVTPRGDWNEGSKPISINYRTEQKLISEEDL
GGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA
VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIF
Date Re9ue/Date Received 2021-03-05 33 KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAD
APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP
QRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG
LYQGLSTATKDTYDALHMQALPPR
SEQ ID NO: 38 {YYB 107}
Length: 351 Type: protein Organism: human Sequence:
MGWSCIILFLVATATGVHSVSDVPRDLEVVAATPTSLLISWD
APAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKP
GVDYTITVYAVTPRGDWNEGSKPISINYRTEQKLISEEDLGG
GPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH
TRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQ
PFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP
AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQ
RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL
YQGLSTATKDTYDALHMQALPPR
SEQ ID NO: 39 {YYB 105}
Length: 497 Type: protein Organism: human Sequence:
MGWSCIILFLVATATGVHSQVQLQESGGGLVKPGGSLKLSC
AASGFTFSKFGMSWVRQTPDKRLEWVASISTGGYNTFYSD
NVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARGYSST
SFAMDYWGQGTMVTVSSGSTSGSGKPGSGEGSDIQMTQSP
SSLSASVGDRVTITCMTSTDIDDDMNWYQQKPGKTPKLLIY
EGNTLRPGVPSRFSGSGSGTDFIFTISSLQPEDIATYYCLQS
FNVPLTFGGGTKVEIKEQKLISEEDLGGGPRKPTTTPAPRPP
TPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFALAYLLDGI
LFIYGVILTALFLRVKRGRKKLLYIFKQPFMRPVQTTQEEDG
CSCRFPEEEEGGCELKFSRSADAPAYQQGQNQLYNELNLG
RREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK
MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM
QALPPR
SEQ ID NO: 40 {T2A peptide}
Length: 21 Type: protein Organism: human Sequence:
Date Recue/Date Received 2021-03-05 34 GSGEGRGSLLTCGDVEENPGP
SEQ ID NO: 41 {P2A peptide}
Length: 22 Type: protein Organism: human Sequence:
GSGATNFSLLKQAGDVEENPGP
SEQ ID NO: 42 {E2A peptide}
Length: 23 Type: protein Organism: human Sequence:
GSGQCTNYALLKLAGDVESNPGP
SEQ ID NO: 43 {F2A peptide}
Length: 25 Type: protein Organism: human Sequence:
GSGVKQTLNFDLLKLAGDVESNPGP
Date Recue/Date Received 2021-03-05 35
Claims (49)
- [Claim 1] A polypeptide comprising a chimeric antigen receptor, which comprises an antigen binding domain; a hinge region; a transmembrane domain; a costimulatory domain; and a signaling domain, wherein the signaling domain comprises a CD3 domain.
- [Claim 2] The polypeptide of claim 1, which comprises IL-7Ra or a part thereof interposed between the costimulatory domain and the CD3 domain.
- [Claim 3] The polypeptide of claim 1, which comprises IL-2R[3 or a part thereof interposed between the costimulatory domain and the CD3 domain.
- [Claim 4] The polypeptide of claim 2, wherein a part of IL-7Ra is a sequence of SEQ
ID NO: 15. - [Claim 5] The polypeptide of claim 3, wherein a part of IL-2R[3 is a sequence of SEQ
ID NO: 17. - [Claim 6] The polypeptide of claim 2, which comprises three immunoreceptor tyrosine-based activation motifs (ITAMs) within the CD3 domain, wherein in a third ITAM region, a first motif, YxxL, is substituted with YxxQ, and a second motif region, YxxLHM, is substituted with YxYVTM.
- [Claim 7] The polypeptide of claim 3, which comprises three immunoreceptor tyrosine-based activation motifs (ITAMs) interposed within the CD3 domain, wherein in a third ITAM region, a first motif, YxxL, is substituted with YyyL, and a second motif, YxxL, is substituted with YxxQ.
- [Claim 8] The polypeptide of claim 4, which comprises three immunoreceptor tyrosine-based activation motifs (ITAMs) within the CD3 domain, wherein a third ITAM region is mutated to a sequence of SEQ ID NO: 31.
- [Claim 9] The polypeptide of claim 5, which comprises three immunoreceptor tyrosine-based activation motifs (ITAMs) within the CD3 domain, wherein a third ITAM region is mutated to a sequence of SEQ ID NO: 32.
- [Claim 10] The polypeptide of claim 4 or 5, wherein the CD3 domain is a sequence represented by SEQ ID NO: 18.
- [Claim 11] The polypeptide of claim 4 or 5, wherein a third ITAM region is a sequence between the 91st and the 113th positions in a sequence represented by SEQ ID
NO: 18. - [Claim 12] The polypeptide of claim 4, wherein in a third ITAM region, a first motif, Date Recue/Date Received 2021-03-05 36 YxxL, is substituted with YxxQ, and a second motif, YxxLHM, is substituted with YxYVTM, wherein x is any amino acid.
- [Claim 13] The polypeptide of claim 5, wherein in a third ITAM region, a first motif, YxxL, is substituted with YyyL, and a second motif, YxxL, is substituted with YxxQ, wherein x and y are each any amino acid.
- [Claim 14] The polypeptide of any one of claims 2 to 7, which further comprises a cytokine.
- [Claim 15] The polypeptide of claim 14, wherein the cytokine is IL-21.
- [Claim 16] The polypeptide of claim 15, wherein the cytokine is linked to a chimeric antigen receptor by a self-cleaving peptide.
- [Claim 17] The polypeptide of claim 14, wherein the cytokine expressed in a T cell is separated from the chimeric antigen derivative and then released to the outside of the T
cell. - [Claim 18] The polypeptide of any one of claims 2 to 7, which further comprises a TGF-pR2 exodomain and an IL18R endodomain, wherein an IL18R transmembrane domain is comprised between the TGF-pR2 exodomain and the IL18R endodomain.
- [Claim 19] The polypeptide of claim 18, wherein the TGF-pR2 exodomain is linked to a chimeric antigen receptor by a self-cleaving peptide.
- [Claim 20] The polypeptide of claim 19, wherein the TGF-p exodomain, IL18R
transmembrane domain, and IL18R endodomain, which is expressed in a T cell, is separated from the chimeric antigen receptor, and among these, the TGF-pR2 domain is exposed to the outside of the T cell, wherein the IL18R endodomain is activated by the linking of the TGF-p present outside of the T cell to the TGF-p exodomain. - [Claim 21] The polypeptide of claim 18, which further comprises a cytokine.
- [Claim 22] The polypeptide of claim 21, wherein the cytokine is IL-21.
- [Claim 23] The polypeptide of claim 21, wherein the cytokine is linked to an IL18R
endodomain by a self-cleaving peptide. - [Claim 24] The polypeptide of claim 23, wherein the cytokine expressed in a T cell is separated from the IL18R endodomain and then released to the outside of the T
cell. - [Claim 25] The polypeptide of claim 2 or 3, wherein the antigen binding domain binds Date Recue/Date Received 2021-03-05 37 to an antigen selected from the group consisting of IL13Ra2, an antigen associated with an angiogenesis activity, EGFRvIll, EphA2, aV[33, mesothelin, and glypican.
- [Claim 26] The polypeptide of claim 1, wherein the costimulatory domain comprises one or more selected from the group consisting of 4-1BB and a 0D28 domain.
- [Claim 27] A polypeptide represented by any one sequence of SEQ ID NOS: 23 to and 34 to 37.
- [Claim 28] The polypeptide of any one of claims 1 to 26 for the treatment of solid cancer.
- [Claim 29] A CAR-T cell, wherein a polypeptide comprising the chimeric antigen receptor according to any one of claims 1 to 26 is expressed.
- [Claim 30] A CAR-T cell, wherein a polypeptide comprising the chimeric antigen receptor according to claim 27 is expressed.
- [Claim 31] A CAR expression vector, which is a vector expressing a chimeric antigen receptor (CAR) and which comprises an antigen binding domain; a hinge region;
a transmembrane domain; a costimulatory domain; and a cytoplasmic signaling domain, wherein the CAR expression vector comprises a nucleic acid encoding a CAR, wherein the nucleic acid encoding a CAR comprises a nucleic acid encoding the costimulatory domain and a nucleic acid encoding a CD3 domain as the signaling domain, and which further comprises a nucleic acid encoding IL-7Ra or a part thereof that is interposed between the nucleic acid encoding the costimulatory domain and the nucleic acid encoding the CD3 domain. - [Claim 32] A CAR expression vector, which is a vector expressing a chimeric antigen receptor (CAR) and which comprises an antigen binding domain; a hinge region;
a transmembrane domain; a costimulatory domain; and a cytoplasmic signaling domain, wherein the CAR expression vector comprises a nucleic acid encoding a CAR, wherein the nucleic acid encoding a CAR comprises a nucleic acid encoding the costimulatory domain and a nucleic acid encoding a CD3 domain as the signaling domain, and which further comprises a nucleic acid encoding IL-2R[3 or a part thereof that is interposed between the nucleic acid encoding the costimulatory domain and the nucleic acid encoding the CD3 domain. - [Claim 33] The CAR expression vector of claim 31, wherein a part of IL-7Ra is a sequence of SEQ ID NO: 15.
- [Claim 34] The CAR expression vector of claim 32, wherein a part of IL-2Rp is a sequence of SEQ ID NO: 17.
Date Recue/Date Received 2021-03-05 38 - [Claim 35] The CAR expression vector of claim 31, wherein the nucleic acid encoding the CD3 domain is a nucleic acid encoding a CD3 domain, in which in a third ITAM
region among the three ITAMs present within the CD3 domain, a first motif, YxxL, and a second motif, YxxLHM, are substituted. - [Claim 36] The CAR expression vector of claim 32, wherein the nucleic acid encoding the CD3 domain is a nucleic acid encoding a CD3 domain, in which in a third ITAM
region among the three ITAMs present within the CD3 domain, a first motif, YxxL, and a second motif, YxxL, are substituted. - [Claim 37] The CAR expression vector of claim 35, wherein the nucleic acid encoding the CD3 domain is a nucleic acid encoding a CD3 domain, in which in a third ITAM
region, a first motif, YxxL, is substituted with YxxQ, and a second motif region, YxxLHM, is substituted with YxYVTM, wherein x is any amino acid. - [Claim 38] The CAR expression vector of claim 36, wherein the nucleic acid encoding the CD3 domain is a nucleic acid encoding a CD3 domain, in which in a third ITAM
region, a first motif, YxxL, is substituted with YyyL, and a second motif, YxxL, is substituted with YxxQ, wherein x and y are each any amino acid. - [Claim 39] The CAR expression vector of any one of claims 31 to 36, which further comprises a nucleic acid encoding cytokine IL-21.
- [Claim 40] The CAR expression vector of claim 39, wherein the nucleic acid encoding cytokine IL-21 is linked to a nucleic acid encoding a CAR through a nucleic acid encoding a self-cleaving peptide.
- [Claim 41] The CAR expression vector of any one of claims 31 to 36, which further comprises a nucleic acid encoding a TGF-61R2 exodomain and a nucleic acid encoding an IL18R transmembrane domain and an IL18R endodomain.
- [Claim 42] The CAR expression vector of claim 41, wherein the nucleic acid encoding a TGF-8R2 exodomain is linked to a nucleic acid encoding a CAR through a nucleic acid encoding a self-cleaving peptide.
- [Claim 43] The CAR expression vector of claim 41, which further comprises a nucleic acid encoding cytokine IL-21.
- [Claim 44] The CAR expression vector of claim 43, wherein the nucleic acid encoding cytokine IL-21 is linked to a nucleic acid encoding IL18R endodomain through a self-cleaving peptide-encoding nucleic acid.
- [Claim 45] A CAR-T cell, which is prepared by introducing the vector according to any one of claims 31 to 39.
Date Recue/Date Received 2021-03-05 39 - [Claim 46] A CAR-T cell, which is prepared by introducing the vector according to claim 41.
- [Claim 47] A CAR-T cell, which is prepared by introducing the vector according to claim 43.
- [Claim 48] An anticancer agent comprising the CAR-T cell according to claim 45.
- [Claim 49] The CAR expression vector of claim 37 or 38, wherein the costimulatory domain is one or more selected from the group consisting of 4-1BB and a 0D28 domain.
Date Recue/Date Received 2021-03-05 40
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| US201862727254P | 2018-09-05 | 2018-09-05 | |
| US62/727,254 | 2018-09-05 | ||
| PCT/KR2019/011516 WO2020050667A1 (en) | 2018-09-05 | 2019-09-05 | Chimeric antigen receptor for solid cancer and t cells expressing chimeric antigen receptor |
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| CA (1) | CA3111978A1 (en) |
| IL (1) | IL281232A (en) |
| WO (1) | WO2020050667A1 (en) |
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| GB202012181D0 (en) * | 2020-08-05 | 2020-09-16 | Autolus Ltd | Chimeric receptor |
| CN115989241A (en) * | 2020-08-19 | 2023-04-18 | 山东博安生物技术股份有限公司 | Invisible chimeric antigen receptors and their use to reduce cytotoxicity to normal cells |
| GB202115329D0 (en) * | 2021-10-25 | 2021-12-08 | Autolus Ltd | Chimeric cytokine receptor |
| CN116178562A (en) * | 2021-11-29 | 2023-05-30 | 四川大学华西医院 | Preparation and application of chimeric antigen receptor immune cells constructed based on EFNA1 |
| WO2023164646A2 (en) * | 2022-02-25 | 2023-08-31 | The Regents Of The University Of California | Methods and compositions for treating cancer |
| KR102532266B1 (en) * | 2022-05-31 | 2023-05-15 | 주식회사 유틸렉스 | Chimeric antigen receptors targeting epidermal growth factor receptor |
| KR102532260B1 (en) * | 2022-07-07 | 2023-05-16 | 주식회사 유틸렉스 | Chimeric antigen receptors simultaneously targeting mutant epidermal growth factor receptor and EphA2 |
| WO2024010119A1 (en) * | 2022-07-07 | 2024-01-11 | 주식회사 유틸렉스 | Chimeric antigen receptor simultaneously targeting mutant egfr and epha2 |
| CN115820698A (en) * | 2022-11-22 | 2023-03-21 | 浙江康佰裕生物科技有限公司 | Fusion gene containing chimeric antigen receptor and chimeric switch receptor coding gene and application thereof |
| CN117586425B (en) * | 2024-01-19 | 2024-06-14 | 北京安百胜生物科技有限公司 | Recombinant respiratory syncytial virus particle antigen, preparation method and application thereof |
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| EP3256496B1 (en) * | 2015-02-12 | 2020-12-30 | University Health Network | Chimeric antigen receptors |
| AU2016255535A1 (en) * | 2015-04-30 | 2017-11-16 | University Of Southern California | Secretory TNT CAR cell immunotherapy |
| JP6751493B2 (en) * | 2015-08-05 | 2020-09-09 | セルレブメド インコーポレーテッド | Chimeric antigen receptor and T cells expressing the chimeric antigen receptor |
| DK3541833T3 (en) * | 2016-11-17 | 2024-04-02 | 2Seventy Bio Inc | TGFß signal converter |
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| JP2023134640A (en) | 2023-09-27 |
| KR20210049916A (en) | 2021-05-06 |
| EP3848387A1 (en) | 2021-07-14 |
| US20230056345A1 (en) | 2023-02-23 |
| EP3848387A4 (en) | 2022-06-22 |
| US20210252069A1 (en) | 2021-08-19 |
| BR112021004289A2 (en) | 2021-08-03 |
| JP2021536266A (en) | 2021-12-27 |
| US20230055761A1 (en) | 2023-02-23 |
| KR20240046644A (en) | 2024-04-09 |
| KR102663253B1 (en) | 2024-05-23 |
| US20220387503A1 (en) | 2022-12-08 |
| IL281232A (en) | 2021-04-29 |
| CN113272318A (en) | 2021-08-17 |
| WO2020050667A1 (en) | 2020-03-12 |
| AU2019336031A1 (en) | 2021-04-15 |
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